Tubulysin compounds, methods of making and use

ABSTRACT

Tubulysin compounds of the formula (I) 
                         
where R 1 , R 2 , R 3a , R 3b , R 4 , R 5 , W, and n are as defined herein, are anti-mitotic agents that can be used in the treatment of cancer, especially when conjugated to a targeting moiety.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/764,825, filed Feb. 14, 2013, thedisclosure of which is incorporated herein by reference.

SEQUENCE LISTING

Incorporated herein by reference in its entirety is a Sequence Listingnamed “SEQT_(—)12026USNP.txt,” comprising SEQ ID NO:1 through SEQ IDNO:26, which include nucleic acid and/or amino acid sequences disclosedherein. The Sequence Listing has been submitted herewith in ASCII textformat via EFS-Web, and thus constitutes both the paper and computerreadable form thereof. The Sequence Listing was first created usingPatentIn 3.5 on Jan. 4, 2014, and is approximately 15 KB in size.

BACKGROUND OF THE INVENTION

This invention relates to compounds structurally similar to thetubulysins, conjugates thereof with a ligand, methods for making andusing such compounds and conjugates, and compositions comprising suchcompounds and conjugates.

The tubulysins are cytotoxins originally isolated from cultures of themyxobacteria Archangium gephyra or Angiococcus disciformis, with eachorganism producing a different mixture of tubulysins (Sasse et al. 2000;Reichenbach et al. 1998). Their crystal structure and biosyntheticpathway have been elucidated (Steinmetz et al. 2004) and theirbiosynthesis genes have been sequenced (Hoefle et al. 2006b).Pretubulysin, a biosynthetic precursor of the tubulysins, also has beenshown to possess some activity (Ullrich et al. 2009). (Full citationsfor the documents cited herein by first author or inventor and year arelisted at the end of this specification.)

The tubulysins belong to a group of naturally occurring antimitoticpolypeptides and depsipeptides that includes the phomopsins, thedolastatins, and the cryptophycins (Hamel 2002). Antimitotic agentsother than polypeptides or depsipeptides also exist, for examplepaclitaxel, the maytansines, and the epothilones. During mitosis, acell's microtubules reorganize to form the mitotic spindle, a processrequiring the rapid assembly and disassembly of the microtubuleconstituent proteins α- and β-tubulin. Antimitotic agents block thisprocess and prevent a cell from undergoing mitosis. At the molecularlevel the exact blockage mechanism may differ from one anti-mitoticagent to another. The tubulysins prevent the assembly of the tubulinsinto microtubules, causing the affected cells to accumulate in the G₂/Mphase and undergo apoptosis (Khalil et al. 2006). Paclitaxel effects thesame end result by binding to microtubules and preventing theirdisassembly.

The tubulysins have a tetrapeptidyl scaffold constructed from oneproteinogenic and three non-proteinogenic amino acid subunits as shownin formula (A): N-methylpipecolinic acid (Mep), isoleucine (Ile),tubuvaline (Tuv), and either tubuphenylalanine (Tup, R′ equals H) ortubutyrosine (Tut, R′ equals OH). Among the better-known naturallyoccurring tubulysins (designated A, B, etc.), the sites of structuralvariation are at residues R′, R″ and R′″ of formula (A), as shown inTable 1:

TABLE 1 Naturally Occurring Tubulysins Tubulysin R′ R″ R′″ A OH OC(═O)MeCH₂OC(═O)i-Bu B OH OC(═O)Me CH₂OC(═O)n-Pr C OH OC(═O)Me CH₂OC(═O)Et D HOC(═O)Me CH₂OC(═O)i-Bu E H OC(═O)Me CH₂OC(═O)n-Pr F H OC(═O)MeCH₂OC(═O)Et G OH OC(═O)Me CH₂OC(═O)CH═CH₂ H H OC(═O)Me CH₂OC(═O)Me I OHOC(═O)Me CH₂OC(═O)Me U H OC(═O)Me H V H OH H Y OH OC(═O)Me H Z OH OH HPretubulysin H H Me

Additionally, other naturally occurring tubulysins have been identified(Chai et al. 2010).

Kaur et al. 2006 studied the antiproliferative properties of tubulysin Aand found that it was more potent than other antimitotic agents such aspaclitaxel and vinblastine and was active in xenograft assays against avariety of cancer cell lines. Further, tubulysin A induced apoptosis incancer cells but not normal cells and showed significant potentialanti-angiogenic properties in in vitro assays. The antimitoticproperties of other tubulysins also have been evaluated and generallyhave been found to compare favorably against those of non-tubulysinantimitotic agents (see, e.g., Balasubramanian et al. 2009; Steinmetz etal. 2004; Wipf et al. 2004). For these reasons, there is considerableinterest in the tubulysins as anti-cancer agents (see, e.g., Domling etal. 2005c; Hamel 2002).

Numerous publications describe efforts directed at the synthesis oftubulysins, including: Balasubramanian et al. 2009; Domling et al. 2006;Hoefle et al. 2003; Neri et al. 2006; Peltier et al. 2006; Sani et al.2007; Sasse et al. 2007; Shankar et al. 2009; Shibue et al. 2009 and2010; and Wipf et al. 2004. Other publications describestructure-activity relationship (SAR) studies, via the preparation andevaluation of tubulysin analogs or derivatives: Balasubramanian et al.2008 and 2009; Chai et al. 2011; Domling 2006; Domling et al. 2005a;Ellman et al. 2013; Hoefle et al. 2001 & 2006a; Pando et al. 2011;Patterson et al. 2007 & 2008; Richter 2012a, 2012b, and 2012c; Shankaret al. 2013; Shibue et al. 2011; Sreejith et al. 2011; Vlahov et al.2010a; Wang et al. 2007; Wipf et al. 2007 and 2010; and Zanda et al.2013. The SAR studies mainly explored structural variations in the Mepring, residues R″ and R′″ of the Tuv subunit, and the aromatic ring oraliphatic carbon chain of the Tup/Tut subunit.

Domling et al. 2005 disclose conjugates of tubulysins with a partnermolecule generically described as a polymer or a biomolecule, but withexamples limited to polyethylene glycol (PEG) as the partner molecule.Cheng et al. 2011 also disclose tubulysin analogs adapted for use inconjugates. Other documents disclosing conjugates of tubulysins are Boydet al. 2008 and 2010; Jackson et al. 2013; Vlahov et al. 2008a, 2008band 2010b; Leamon et al. 2008 and 2010; Reddy et al. 2009; and Low etal. 2010. Leung et al. 2002 disclose polyanionic polypeptides that canbe conjugated to drugs (including tubulysins) to improve theirbioactivity and water solubility.

Davis et al. 2008 and Schluep et al. 2009 disclose cyclodextrin basedformulations in which tubulysins are covalently attached to acyclodextrin via a hydrazide-disulfide linker moiety bonded to theTup/Tut carboxyl group.

The deacetylation of the Tuv subunit (i.e., R″ in formula (A) ishydroxyl instead of acetyl) reportedly leads to loss of biologicalactivity (Domling et al. 2006). In a study of tubulysins U and V, whichdiffer in the former being acetylated and the latter being deacetylated,tubulysin V was reported to be less potent by about 200× to 600×,depending on the assay (Balasubramanian et al. 2009). Because an acetategroup is susceptible to hydrolysis, deacetylation at the R″ position isa concern, as a potential instability center leading to loss ofactivity, for the development of tubulysin analogs for pharmaceuticalapplications.

BRIEF SUMMARY OF THE INVENTION

We have discovered that it is possible to guard against the loss ofbiological activity associated with deacetylation as discussed above byreplacing an acetate at the R″ position with a carbamate group. Acarbamate group as described herein does not cause significant loss ofbiological activity but yet is more stable.

Accordingly, in one aspect, this invention provides a compound having astructure represented by formula (I)

wherein

-   -   R¹ is H, unsubstituted or substituted C₁-C₁₀ alkyl,        unsubstituted or substituted C₂-C₁₀ alkenyl, unsubstituted or        substituted C₂-C₁₀ alkynyl, unsubstituted or substituted aryl,        unsubstituted or substituted heteroaryl, unsubstituted or        substituted (CH₂)₁₋₂O(C₁-C₁₀ alkyl), unsubstituted or        substituted (CH₂)₁₋₂O(C₂-C₁₀ alkenyl), unsubstituted or        substituted (CH₂)₁₋₂O(C₂-C₁₀ alkynyl), (CH₂)₁₋₂OC(═O)(C₁-C₁₀        alkyl), unsubstituted or substituted (CH₂)₁₋₂OC(═O)(C₂-C₁₀        alkenyl), unsubstituted or substituted (CH₂)₁₋₂OC(═O)(C₂-C₁₀        alkynyl), unsubstituted or substituted C(═O)(C₁-C₁₀ alkyl),        unsubstituted or substituted C(═O)(C₂-C₁₀ alkenyl),        unsubstituted or substituted C(═O)(C₂-C₁₀ alkynyl),        unsubstituted or substituted cycloaliphatic, unsubstituted or        substituted heterocycloaliphatic, unsubstituted or substituted        arylalkyl, or unsubstituted or substituted alkylaryl;    -   R² is H, unsubstituted or substituted C₁-C₁₀ alkyl,        unsubstituted or substituted C₂-C₁₀ alkenyl, unsubstituted or        substituted C₂-C₁₀ alkynyl, unsubstituted or substituted aryl,        unsubstituted or substituted heteroaryl, unsubstituted or        substituted (CH₂)₁₋₂O(C₁-C₁₀ alkyl), unsubstituted or        substituted (CH₂)₁₋₂O(C₂-C₁₀ alkenyl), unsubstituted or        substituted (CH₂)₁₋₂O(C₂-C₁₀ alkynyl), (CH₂)₁₋₂OC(═O)(C₁-C₁₀        alkyl), unsubstituted or substituted (CH₂)₁₋₂OC(═O)(C₂-C₁₀        alkenyl), unsubstituted or substituted (CH₂)₁₋₂OC(═O)(C₂-C₁₀        alkynyl), unsubstituted or substituted C(═O)(C₁-C₁₀ alkyl),        unsubstituted or substituted C(═O)(C₂-C₁₀ alkenyl),        unsubstituted or substituted C(═O)(C₂-C₁₀ alkynyl),        unsubstituted or substituted cycloaliphatic, unsubstituted or        substituted heterocycloaliphatic, unsubstituted or substituted        arylalkyl, unsubstituted or substituted alkylaryl, or

-   -   -   wherein each R^(2a) is independently H, NH₂, NHMe, Cl, F,            Me, Et, or CN;

    -   R^(3a) and R^(3b) are independently H, C₁-C₅ alkyl, CH₂(C₅-C₆        cycloalkyl), CH₂C₆H₅, C₆H₅, or CH₂CH₂OH;

    -   R⁴ is

-   -   -   wherein R^(4a) is H or C₁-C₃ alkyl; and Y is H, OH, Cl, F,            CN, Me, Et, NO₂, or NH₂;

    -   R⁵ is H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl, CO(C₁-C₅        alkyl), CO(C₂-C₅ alkenyl), or CO(C₂-C₅ alkynyl);

    -   W is O or S (preferably O); and

    -   n is 0, 1, or 2;        or a pharmaceutically acceptable salt thereof

In another embodiment, this invention provides a conjugate comprising acompound according to formula (I) covalently linked to a targetingmoiety that specifically or preferentially binds to a chemical entity ona target cell, which target cell preferably is a cancer cell.Preferably, the targeting moiety is an antibody—more preferably amonoclonal antibody and even more preferably a human monoclonalantibody—or the antigen-binding portion thereof and the chemical entityis a tumor associated antigen. the tumor associated antigen can be onethat is displayed on the surface of a cancer cell or one that issecreted by a cancer cell into the surrounding extracellular space.

In another embodiment, there is provided a composition of mattercomprising a compound of this invention and a linker moiety having areactive functional group, suitable for conjugation to a targetingmoiety.

In another embodiment, there is provided a method of treating a cancerin a subject suffering from such cancer, comprising administering to thesubject a therapeutically effective amount of a compound of thisinvention or a conjugate thereof with a targeting moiety (particularlyan antibody). In another embodiment, there is provided the use of acompound of this invention or a conjugate thereof with a targetingmoiety (particularly an antibody) for the preparation of a medicamentfor the treatment of cancer in a subject suffering from such cancer. Thecancer can be renal, gastric, lung, or ovarian cancer.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIGS. 1, 2 a-2 b, and 3 show, in combination, a scheme for the synthesisof compound (III-1).

FIG. 4 shows a scheme for the synthesis of compound (III-2).

FIG. 5 shows a scheme for the synthesis of compounds (I-2) and (I-3).

FIGS. 6 a-6 c show in combination a scheme for the synthesis ofcompounds (III-4) and (III-5).

FIG. 7 shows a scheme for the synthesis of compound (I-1).

FIG. 8 shows a scheme for the synthesis of compound (I-4).

FIGS. 9 a and 9 b show in combination a scheme for the synthesis ofcompound (III-6).

FIGS. 10 and 11 show schemes for the synthesis of intermediates usefulfor making compounds of this invention.

FIG. 12 shows schemes for synthesis of additional compounds of thisinvention.

FIGS. 13 a-13 d show the biological activity of some compounds of thisinvention.

FIG. 14 shows the in vitro activity of a conjugate of this invention.

FIG. 15 shows the in vivo activity of a conjugate of this invention.

FIGS. 16 a, 16 b, 17 a, 17 b, 18 a, 18 b, 19 a, 19 b, and 20 presentadditional in vivo data on the activity of conjugates of this invention.

FIGS. 21 and 22 show schemes for the synthesis of additional compoundsof this invention, illustrating structural variations at the carbamategroup.

FIG. 23 shows a scheme for the synthesis of compounds of this inventionsuitable for conjugation via “click” chemistry.

FIG. 24 shows a scheme for the synthesis of compounds of this inventionsuitable for conjugation via an aliphatic amine group.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“Antibody” means whole antibodies and any antigen binding fragment(i.e., “antigen-binding portion”) or single chain variants thereof. Awhole antibody is a protein comprising at least two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds. Each heavychain comprises a heavy chain variable region (V_(H)) and a heavy chainconstant region comprising three domains, C_(H1), C_(H2) and C_(H3).Each light chain comprises a light chain variable region (V_(L) orV_(k)) and a light chain constant region comprising one single domain,C_(L). The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDRs), interspersed with more conserved framework regions (FRs). EachV_(H) and V_(L) comprises three CDRs and four FRs, arranged from amino-to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. The variable regions contain a binding domain thatinteracts with an antigen. The constant regions may mediate the bindingof the antibody to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. An antibody is said to “specificallybind” to an antigen X if the antibody binds to antigen X with a K_(D) of5×10⁻⁸ M or less, more preferably 1×10⁻⁸ M or less, more preferably6×10⁻⁹ M or less, more preferably 3×10⁻⁹ M or less, even more preferably2×10⁻⁹ M or less. The antibody can be chimeric, humanized, or,preferably, human. The heavy chain constant region can be engineered toaffect glycosylation type or extent, to extend antibody half-life, toenhance or reduce interactions with effector cells or the complementsystem, or to modulate some other property. The engineering can beaccomplished by replacement, addition, or deletion of one or more aminoacids or by replacement of a domain with a domain from anotherimmunoglobulin type, or a combination of the foregoing.

“Antigen binding fragment” and “antigen binding portion” of an antibody(or simply “antibody portion” or “antibody fragment”) mean one or morefragments of an antibody that retain the ability to specifically bind toan antigen. It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody, suchas (i) a Fab fragment, a monovalent fragment consisting of the V_(L),V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fab′ fragment, which is essentially an Fabwith part of the hinge region (see, for example, Abbas et al., Cellularand Molecular Immunology, 6th Ed., Saunders Elsevier 2007); (iv) a Fdfragment consisting of the V_(H) and C_(Hi) domains; (v) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546),which consists of a V_(H) domain; (vii) an isolated complementaritydetermining region (CDR); and (viii) a nanobody, a heavy chain variableregion containing a single variable domain and two constant domains.Preferred antigen binding fragments are Fab, F(ab′)₂, Fab′, Fv, and Fdfragments. Furthermore, although the two domains of the Fv fragment,V_(L) and V_(H), are encoded by separate genes, they can be joined,using recombinant methods, by a synthetic linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single chain Fv, or scFv); see,e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodiesare also encompassed within the term “antigen-binding portion” of anantibody.

An “isolated antibody” means an antibody that is substantially free ofother antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds antigen X is substantiallyfree of antibodies that specifically bind antigens other than antigenX). An isolated antibody that specifically binds antigen X may, however,have cross-reactivity to other antigens, such as antigen X moleculesfrom other species. In certain embodiments, an isolated antibodyspecifically binds to human antigen X and does not cross-react withother (non-human) antigen X antigens. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

“Monoclonal antibody” or “monoclonal antibody composition” means apreparation of antibody molecules of single molecular composition, whichdisplays a single binding specificity and affinity for a particularepitope.

“Human antibody” means an antibody having variable regions in which boththe framework and CDR regions (and the constant region, if present) arederived from human germline immunoglobulin sequences. Human antibodiesmay include later modifications, including natural or syntheticmodifications. Human antibodies may include amino acid residues notencoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo). However, “human antibody” does not include antibodiesin which CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

“Human monoclonal antibody” means an antibody displaying a singlebinding specificity, which has variable regions in which both theframework and CDR regions are derived from human germline immunoglobulinsequences. In one embodiment, human monoclonal antibodies are producedby a hybridoma that includes a B cell obtained from a transgenicnonhuman animal, e.g., a transgenic mouse, having a genome comprising ahuman heavy chain transgene and a light chain transgene fused to animmortalized cell.

“Aliphatic” means a straight- or branched-chain, saturated orunsaturated, non-aromatic hydrocarbon moiety having the specified numberof carbon atoms (e.g., as in “C₃ aliphatic,” “C₁-C₅ aliphatic,” or “C₁to C₅ aliphatic,” the latter two phrases being synonymous for analiphatic moiety having from 1 to 5 carbon atoms) or, where the numberof carbon atoms is not explicitly specified, from 1 to 4 carbon atoms (2to 4 carbons in the instance of unsaturated aliphatic moieties).

“Alkyl” means a saturated aliphatic moiety, with the same convention fordesignating the number of carbon atoms being applicable. By way ofillustration, C₁-C₄ alkyl moieties include, but are not limited to,methyl, ethyl, propyl, isopropyl, isobutyl, t-butyl, 1-butyl, 2-butyl,and the like. “Alkylene” means a divalent counterpart of an alkyl group,such as CH₂CH₂, CH₂CH₂CH₂, and CH₂CH₂CH₂CH₂.

“Alkenyl” means an aliphatic moiety having at least one carbon-carbondouble bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkenylmoieties include, but are not limited to, ethenyl (vinyl), 2-propenyl(allyl or prop-2-enyl), cis-1-propenyl, trans-1-propenyl, E- (or Z-)2-butenyl, 3-butenyl, 1,3-butadienyl (but-1,3-dienyl) and the like.

“Alkynyl” means an aliphatic moiety having at least one carbon-carbontriple bond, with the same convention for designating the number ofcarbon atoms being applicable. By way of illustration, C₂-C₄ alkynylgroups include ethynyl (acetylenyl), propargyl (prop-2-ynyl),1-propynyl, but-2-ynyl, and the like.

“Cycloaliphatic” means a saturated or unsaturated, non-aromatichydrocarbon moiety having from 1 to 3 rings, each ring having from 3 to8 (preferably from 3 to 6) carbon atoms. “Cycloalkyl” means acycloaliphatic moiety in which each ring is saturated. “Cycloalkenyl”means a cycloaliphatic moiety in which at least one ring has at leastone carbon-carbon double bond. “Cycloalkynyl” means a cycloaliphaticmoiety in which at least one ring has at least one carbon-carbon triplebond. By way of illustration, cycloaliphatic moieties include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and adamantyl.Preferred cycloaliphatic moieties are cycloalkyl ones, especiallycyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. “Cycloalkylene”means a divalent counterpart of a cycloalkyl group.

“Heterocycloaliphatic” means a cycloaliphatic moiety wherein, in atleast one ring thereof, up to three (preferably 1 to 2) carbons havebeen replaced with a heteroatom independently selected from N, O, or S,where the N and S optionally may be oxidized and the N optionally may bequaternized. Similarly, “heterocycloalkyl,” “heterocycloalkenyl,” and“heterocycloalkynyl” means a cycloalkyl, cycloalkenyl, or cycloalkynylmoiety, respectively, in which at least one ring thereof has been somodified. Exemplary heterocycloaliphatic moieties include aziridinyl,azetidinyl, 1,3-dioxanyl, oxetanyl, tetrahydrofuryl, pyrrolidinyl,piperidinyl, piperazinyl, tetrahydropyranyl, tetrahydrothiopyranyl,tetrahydrothiopyranyl sulfone, morpholinyl, thiomorpholinyl,thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolanyl,tetrahydro-1,1-dioxothienyl, 1,4-dioxanyl, thietanyl, and the like.“Heterocycloalkylene” means a divalent counterpart of a heterocycloalkylgroup.

“Alkoxy,” “aryloxy,” “alkylthio,” and “arylthio” mean —O(alkyl),—O(aryl), —S(alkyl), and —S(aryl), respectively. Examples are methoxy,phenoxy, methylthio, and phenylthio, respectively.

“Halogen” or “halo” means fluorine, chlorine, bromine or iodine.

“Aryl” means a hydrocarbon moiety having a mono-, bi-, or tricyclic ringsystem wherein each ring has from 3 to 7 carbon atoms and at least onering is aromatic. The rings in the ring system may be fused to eachother (as in naphthyl) or bonded to each other (as in biphenyl) and maybe fused or bonded to non-aromatic rings (as in indanyl orcyclohexyl-phenyl). By way of further illustration, aryl moietiesinclude, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl,indanyl, biphenyl, phenanthryl, anthracenyl, and acenaphthyl. “Arylene”means a divalent counterpart of an aryl group, for example1,2-phenylene, 1,3-phenylene, or 1,4-phenylene.

“Heteroaryl” means a moiety having a mono-, bi-, or tricyclic ringsystem wherein each ring has from 3 to 7 carbon atoms and at least onering is an aromatic ring containing from 1 to 4 heteroatomsindependently selected from N, O, or S, where the N and S optionally maybe oxidized and the N optionally may be quaternized. Such at least oneheteroatom containing aromatic ring may be fused to other types of rings(as in benzofuranyl or tetrahydroisoquinolyl) or directly bonded toother types of rings (as in phenylpyridyl or 2-cyclopentylpyridyl). Byway of further illustration, heteroaryl moieties include pyrrolyl,furanyl, thiophenyl (thienyl), imidazolyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, pyridyl,N-oxopyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, quinolinyl,isoquinolynyl, quinazolinyl, cinnolinyl, quinozalinyl, naphthyridinyl,benzofuranyl, indolyl, benzothiophenyl, oxadiazolyl, thiadiazolyl,phenothiazolyl, benzimidazolyl, benzotriazolyl, dibenzofuranyl,carbazolyl, dibenzothiophenyl, acridinyl, and the like. “Heteroarylene”means a divalent counterpart of an aryl group.

Where it is indicated that a moiety may be substituted, such as by useof “unsubstituted or substituted” or “optionally substituted” phrasingas in “unsubstituted or substituted C₁-C₅ alkyl” or “optionallysubstituted heteroaryl,” such moiety may have one or more independentlyselected substituents, preferably one to five in number, more preferablyone or two in number. Substituents and substitution patterns can beselected by one of ordinary skill in the art, having regard for themoiety to which the substituent is attached, to provide compounds thatare chemically stable and that can be synthesized by techniques known inthe art as well as the methods set forth herein.

“Arylalkyl,” (heterocycloaliphatic)alkyl,” “arylalkenyl,” “arylalkynyl,”“biarylalkyl,” and the like mean an alkyl, alkenyl, or alkynyl moiety,as the case may be, substituted with an aryl, heterocycloaliphatic,biaryl, etc., moiety, as the case may be, with the open (unsatisfied)valence at the alkyl, alkenyl, or alkynyl moiety, for example as inbenzyl, phenethyl, N-imidazoylethyl, N-morpholinoethyl, and the like.Conversely, “alkylaryl,” “alkenylcycloalkyl,” and the like mean an aryl,cycloalkyl, etc., moiety, as the case may be, substituted with an alkyl,alkenyl, etc., moiety, as the case may be, for example as inmethylphenyl (tolyl) or allylcyclohexyl. “Hydroxyalkyl,” “haloalkyl,”“alkylaryl,” “cyanoaryl,” and the like mean an alkyl, aryl, etc.,moiety, as the case may be, substituted with one or more of theidentified substituent (hydroxyl, halo, etc., as the case may be).

For example, permissible substituents include, but are not limited to,alkyl (especially methyl or ethyl), alkenyl (especially allyl), alkynyl,aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic, halo (especiallyfluoro), haloalkyl (especially trifluoromethyl), hydroxyl, hydroxyalkyl(especially hydroxyethyl), cyano, nitro, alkoxy, —O(hydroxyalkyl),—O(haloalkyl) (especially —OCF₃), —O(cycloalkyl), —O(heterocycloalkyl),—O(aryl), alkylthio, arylthio, ═O, ═NH, ═N(alkyl), ═NOH, ═NO(alkyl),—C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH, —CO(═O)(alkyl),—CO(═O)(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)(alkyl),—OC(═O)(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(cylcoalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl),—SO₂N(alkyl)₂, and the like.

Where the moiety being substituted is an aliphatic moiety, preferredsubstituents are aryl, heteroaryl, cycloaliphatic, heterocycloaliphatic,halo, hydroxyl, cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl),—O(cycloalkyl), —O(heterocycloalkyl), —O(aryl), alkylthio, arylthio, ═O,═NH, ═N(alkyl), ═NOH, ═NO(alkyl), —CO₂H, —C(═O)NHOH, —CO(═O)(alkyl),—CO(═O)(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)(alkyl),—OC(═O)(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —S(alkyl), —S(aryl),—S(═O)alkyl, —S(cycloalkyl), —SO₂(alkyl), —SO₂NH₂, —SO₂NH(alkyl), and—SO₂N(alkyl)₂. More preferred substituents are halo, hydroxyl, cyano,nitro, alkoxy, —O(aryl), ═O, ═NOH, ═NO(alkyl), —OC(═O)(alkyl),—OC(═O)(alkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂, azido,—NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.Especially preferred are phenyl, cyano, halo, hydroxyl, nitro,C₁-C₄alkyoxy, O(C₂-C₄ alkylene)OH, and O(C₂-C₄ alkylene)halo.

Where the moiety being substituted is a cycloaliphatic,heterocycloaliphatic, aryl, or heteroaryl moiety, preferred substituentsare alkyl, alkenyl, alkynyl, halo, haloalkyl, hydroxyl, hydroxyalkyl,cyano, nitro, alkoxy, —O(hydroxyalkyl), —O(haloalkyl), —O(aryl),—O(cycloalkyl), —O(heterocycloalkyl), alkylthio, arylthio,—C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH, —CO(═O)(alkyl),—CO(═O)(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl), —C(═O)N(alkyl)₂,—OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)(alkyl),—OC(═O)(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,azido, —NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NH(hydroxyalkyl),—NHC(═O)(alkyl), —NHC(═O)H, —NHC(═O)NH₂, —NHC(═O)NH(alkyl),—NHC(═O)N(alkyl)₂, —NHC(═NH)NH₂, —OSO₂(alkyl), —SH, —NH(alkyl),—N(alkyl), —NH(hydroxyalkyl), —S(═O)alkyl, —SO₂(alkyl), —SO₂NH₂,—SO₂NH(alkyl), and —SO₂N(alkyl)₂. More preferred substituents are alkyl,alkenyl, halo, haloalkyl, hydroxyl, hydroxyalkyl, cyano, nitro, alkoxy,—O(hydroxyalkyl), —C(═O)(alkyl), —C(═O)H, —CO₂H, —C(═O)NHOH,—CO(═O)(alkyl), —CO(═O)(hydroxyalkyl), —C(═O)NH₂, —C(═O)NH(alkyl),—C(═O)N(alkyl)₂, —OC(═O)(alkyl), —OC(═O)(hydroxyalkyl), —OC(═O)(alkyl),—OC(═O)(hydroxyalkyl), —OC(═O)NH₂, —OC(═O)NH(alkyl), —OC(═O)N(alkyl)₂,—NH₂, —NH(alkyl), —N(alkyl)₂, —NH(aryl), —NHC(═O)(alkyl), —NHC(═O)H,—NHC(═O)NH₂, —NHC(═O)NH(alkyl), —NHC(═O)N(alkyl)₂, and —NHC(═NH)NH₂.Especially preferred are C₁-C₄ alkyl, cyano, nitro, halo, andC₁-C₄alkoxy.

Where a range is stated, as in “C₁-C₅ alkyl” or “5 to 10%,” such rangeincludes the end points of the range, as in C₁ and C₅ in the firstinstance and 5% and 10% in the second instance.

Unless particular stereoisomers are specifically indicated (e.g., by abolded or dashed bond at a relevant stereocenter in a structuralformula, by depiction of a double bond as having E or Z configuration ina structural formula, or by use stereochemistry-designatingnomenclature), all stereoisomers are included within the scope of theinvention, as pure compounds as well as mixtures thereof. Unlessotherwise indicated, individual enantiomers, diastereomers, geometricalisomers, and combinations and mixtures thereof are all encompassed bythis invention.

Those skilled in the art will appreciate that compounds may havetautomeric forms (e.g., keto and enol forms), resonance forms, andzwitterionic forms that are equivalent to those depicted in thestructural formulae used herein and that the structural formulaeencompass such tautomeric, resonance, or zwitterionic forms.

“Pharmaceutically acceptable ester” means an ester that hydrolyzes invivo (for example in the human body) to produce the parent compound or asalt thereof or has per se activity similar to that of the parentcompound. Suitable esters include C₁-C₅ alkyl, C₂-C₅ alkenyl or C₂-C₅alkynyl esters, especially methyl, ethyl or n-propyl.

“Pharmaceutically acceptable salt” means a salt of a compound suitablefor pharmaceutical formulation. Where a compound has one or more basicgroups, the salt can be an acid addition salt, such as a sulfate,hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate,pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate,methylsulfate, fumarate, benzoate, succinate, mesylate, lactobionate,suberate, tosylate, and the like. Where a compound has one or moreacidic groups, the salt can be a salt such as a calcium salt, potassiumsalt, magnesium salt, meglumine salt, ammonium salt, zinc salt,piperazine salt, tromethamine salt, lithium salt, choline salt,diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodiumsalt, tetramethylammonium salt, and the like. Polymorphic crystallineforms and solvates are also encompassed within the scope of thisinvention.

Compositions

In formula (I), repeated below for convenience,

the group R¹ preferably is Me, Et, n-Pr, i-Pr, or

more preferably the latter.

Also in formula (I), the group R² preferably is C₁-C₅ alkyl, C₁-C₅alkenyl, C₁-C₅ alkynyl, CH₂OC(═O)C₁-C₅ alkyl, CH₂OC(═O)C₁-C₅ alkenyl,CH₂OC(═O)C₁-C₅ alkynyl,

Also in formula (I), preferred groups N(R^(3a))(R^(3b)) are:

with it being especially preferred that one of R^(3a) and R^(3b) is Hand the other is Me. In other preferred embodiments, R^(3a) and R^(3b)are both H or both Me, or one of R^(3a) and R^(3b) is H and the other isC₆H₅.

In another preferred embodiment, R^(3a) and R^(3b) are independently H,C₁-C₅ alkyl, CH₂(C₅-C₆ cycloalkyl), CH₂C₆H₅, or CH₂CH₂OH.

In the definitions of R¹ and R² in formula (I), where a group is definedas being either unsubstituted or substituted, it preferably isunsubstituted.

In the formulae of this specification, a bond traversing a phenyl ringbetween two carbons of the phenyl ring means that the group attached tothe bond may be located at any of the ortho, meta, or para positions ofthe phenyl ring. By way of illustration, the formula

represents

The synthesis of counterparts of the tubulysin Tuv and Tup subunits withvarious R² and R⁴ groups is taught by Cheng et al. 2011, the disclosureof which is incorporated herein by reference.

In a preferred embodiment of compounds according to formula (I), R¹ is

-   R₂ is C₁-C₅ alkyl (especially Me or n-Pr) or

-   one of R^(3a) and R^(3b) is H and the other is Me; R⁴ is

where Y is H or NO₂ and R^(4a) is H, Me, or Et;

-   R⁵ is Me; W is O, and n is 1.

In another preferred embodiment of compounds according to formula (I), nis 1, W is O, Y in R⁴ is H or NO₂ (preferably H), and R² is

and more preferably

A compound according to this preferred embodiment is represented byformula (Ia)

wherein Y is H or NO₂; R^(4a) is H, Me, or Et; and R^(3a) and R^(3b) areindependently H, C₆H₅, Me, or Et; or a pharmaceutically acceptable saltthereof

Even more preferably, the compound has a structure represented byformula (Ia′):

where R^(4a) is H, Me, or Et; and R^(3a) and R^(3b) are independently H,C₆H₅, Me, or Et; or a pharmaceutically acceptable salt thereof

In yet another preferred embodiment, W is O, Y is NH₂, n is 1, and bothgroups R^(2a) in R² are other than NH₂. A compound according to thisembodiment is represented by formula (Ib):

where R^(4a) is H, Me, or Et; R^(3a) and R^(3b) are independently H,C₆H₅, Me, and Et; and R⁶ is C₁-C₅ alkyl, CH₂OC(═O)C₁-C₅ alkyl, or(CH₂)₁₋₂C₆H₅; or a pharmaceutically acceptable salt thereof.

In yet another preferred embodiment, the compound has a structurerepresented by formula (Ib′):

where R^(4a) is H, Me, or Et (preferably H) and R⁶ is Me or n-Pr; or apharmaceutically acceptable salt thereof

Preferably, in formulae (Ia), (Ia′), and (Ib), one of R^(3a) and R^(3b)is H and the other is Me. In other preferred embodiments, R^(3a) andR^(3b) are both H or both Me, or one of R^(3a) and R^(3b) is H and theother is C₆H₅. In yet other preferred embodiments, R^(3a) and R^(3b) areindependently H, Me, or Et

Specific examples of compounds of this invention include the compoundsshown immediately following, along with their pharmaceuticallyacceptable salts:

Compounds (I-2), (I-7), (I-8) and (I-9) are preferred.

Conjugates

Optionally, compounds of this invention may be conjugated to a targetingmoiety that specifically or preferentially binds to a chemical entity ona cancer cell. Preferably, the targeting moiety is an antibody orantigen binding portion thereof and the chemical entity is a tumorassociated antigen. Preferably, the conjugation is effected through achemical bond to a functional group in the Tuv or Tup subunit, such asan amino group.

In another embodiment, there is provided a conjugate comprisingcytotoxic compound according to this invention and a ligand, representedby formula (II)[D(X^(D))_(a)C(X^(Z))_(b)]_(m)Z  (II)where Z is a ligand; D is a cytotoxic compound according to thisinvention (e.g., a compound according to formula (I), (Ia), (Ia′), or(Ib)); and —(X^(D))_(a)C(X^(Z))_(b)— are collectively referred to as a“linker moiety” or “linker” because they link Z and D. Within thelinker, C is a cleavable group designed to be cleaved at or near thesite of intended biological action of compound D; X^(D) and X^(Z) arereferred to as spacer moieties (or “spacers”) because they space apart Dand C and C and Z, respectively; subscripts a and b are independently 0or 1 (that is, the presence of X^(D) and/or X^(Z) are optional); andsubscript m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 1, 2, 3, or4). D, X^(D), C, X^(Z) and Z are more fully described hereinbelow.

Ligand Z—for example an antibody—serves a targeting function. By bindingto a target tissue or cell where its antigen or receptor is located,ligand Z directs the conjugate there. Preferably, the target tissue orcell is a cancer tissue or cell and the antigen or receptor is atumor-associated antigen, that is, an antigen that is uniquely expressedby cancerous cells or is overexpressed by cancer cells, compared tonon-cancerous cells. Cleavage of group C at the target tissue or cellreleases compound D to exert its cytotoxic effect locally. In someinstances, the conjugate is internalized into a target cell byendocytosis and cleavage takes place within the target cell. In thismanner, precise delivery of compound D is achieved at the site ofintended action, reducing the dosage needed. Also, compound D isnormally biologically inactive (or significantly less active) in itsconjugated state, thereby reducing undesired toxicity against non-targettissue or cells. As anticancer drugs are often highly toxic to cells ingeneral, this is an important consideration.

As reflected by the subscript m, each molecule of ligand Z can conjugatewith more than one compound D, depending on the number of sites ligand Zhas available for conjugation and the experimental conditions employed.Those skilled in the art will appreciate that, while each individualmolecule of ligand Z is conjugated to an integer number of compounds D,a preparation of the conjugate may analyze for a non-integer ratio ofcompounds D to ligand Z, reflecting a statistical average. This ratio isreferred to as the substitution ratio (SR) or, alternatively, in thecase of antibody-drug conjugates, the drug-antibody ratio (DAR).

Ligand Z and Conjugation Thereof

Preferably, ligand Z is an antibody. For convenience and brevity and notof limitation, the detailed subsequent discussion herein about theconjugation of ligand Z is written in the context of its being anantibody, but those skilled in the art will understand that other typesof ligand Z can be conjugated, mutatis mutandis. For example, conjugateswith folic acid as the ligand can target cells having the folatereceptor on their surfaces (Vlahov et al. 2008; Leamon et al. 2008). Forthe same reason, the detailed discussion below is primarily written interms of a 1:1 ratio of antibody Z to compound D (m=1).

Preferably, ligand Z is an antibody against a tumor associated antigen,allowing a conjugate comprising such a ligand Z to selectively targetcancer cells. Examples of such antigens include: mesothelin, prostatespecific membrane antigen (PSMA), CD19, CD22, CD30, CD70, B7H4 (alsoknown as 08E), protein tyrosine kinase 7 (PTK7), glypican-3, RG1,CTLA-4, and CD44. The antibody can be animal (e.g., murine), chimeric,humanized, or, preferably, human. The antibody preferably is monoclonal,especially a monoclonal human antibody. The preparation of humanmonoclonal antibodies against some of the aforementioned antigens isdisclosed in Korman et al., US 2009/0074660 A1 (B7H4); Rao-Naik et al.,8,097,703 B2 (CD19); King et al., US 2010/0143368 A1 (CD22); Keler etal., U.S. Pat. No. 7,387,776 B2 (2008) (CD30); Terrett et al., U.S. Pat.No. 8,124,738 B2 (CD70); Korman et al., U.S. Pat. No. 6,984,720 B1(2006) (CTLA-4); Korman et al., U.S. Pat. No. 8,008,449 B2 (2011)(PD-1); Huang et al., US 2009/0297438 A1 and Cardarelli et al., U.S.Pat. No. 7,875,278 B2 (PSMA); Terrett et al., US 2010/0034826 A1 (PTK7);Terrett et al., US 2010/0209432 (A1) (glypican-3); Harkins et al., U.S.Pat. No. 7,335,748 B2(2008) (RG1); Terrett et al., U.S. Pat. No.8,268,970 B2 (2012) (mesothelin); and Xu et al., US 2010/0092484 A1(CD44); the disclosures of which are incorporated herein by reference.

Ligand Z can also be an antibody fragment or antibody mimetic, such asan affibody, a domain antibody (dAb), a nanobody, a unibody, a DARPin,an anticalin, a versabody, a duocalin, a lipocalin, or an avimer.

Any one of several different reactive groups on ligand Z can be aconjugation site, including ε-amino groups in lysine residues, pendantcarbohydrate moieties, carboxylic acid groups, disulfide groups, andthiol groups. Each type of reactive group represents a trade-off, havingsome advantages and some disadvantages. For reviews on antibody reactivegroups suitable for conjugation, see, e.g., Garnett, Adv. Drug DeliveryRev. 53 (2001), 171-216 and Dubowchik and Walker, Pharmacology &Therapeutics 83 (1999), 67-123, the disclosures of which areincorporated herein by reference.

In one embodiment, ligand Z is conjugated via a lysine ε-amino group.Most antibodies have multiple exposed lysine ε-amino groups, which canbe conjugated via amide, urea, thiourea, or carbamate bonds usingtechniques known in the art, including modification with aheterobifunctional agent (as further described hereinbelow). However, itis difficult to control which and how many ε-amino groups react, leadingto potential batch-to-batch variability in conjugate preparations. Also,conjugation may cause neutralization of a protonated ε-amino groupimportant for maintaining the antibody's native conformation or may takeplace at a lysine near or at the antigen binding site, neither being adesirable occurrence.

In another embodiment, ligand Z can be conjugated via a carbohydrateside chain, as many antibodies are glycosylated. The carbohydrate sidechain can be oxidized with periodate to generate aldehyde groups, whichin turn can be reacted with amines to form an imine group, such as in asemicarbazone, oxime, or hydrazone. If desired, the imine group can beconverted to a more stable amine group by reduction with sodiumcyanoborohydride. For additional disclosures on conjugation viacarbohydrate side chains, see, e.g., Rodwell et al., Proc. Nat'l Acad.Sci. USA 83, 2632-2636 (1986); the disclosure of which is incorporatedherein by reference. As with lysine ε-amino groups, there are concernsregarding reproducibility of the location of the conjugation site(s) andstoichiometry.

In yet another embodiment, ligand Z can be conjugated via a carboxylicacid group. In one embodiment, a terminal carboxylic acid group isfunctionalized to generate a carbohydrazide, which is then reacted withan aldehyde-bearing conjugation moiety. See Fisch et al., BioconjugateChemistry 1992, 3, 147-153.

In yet another embodiment, antibody Z can be conjugated via a disulfidegroup bridging a cysteine residue on antibody Z and a sulfur on theother portion of the conjugate. Some antibodies lack free thiol(sulfhydryl) groups but have disulfide groups, for example in the hingeregion. In such case, free thiol groups can be generated by reduction ofnative disulfide groups. The thiol groups so generated can then be usedfor conjugation. See, e.g., Packard et al., Biochemistry 1986, 25,3548-3552; King et al., Cancer Res. 54, 6176-6185 (1994); and Doroninaet al., Nature Biotechnol. 21(7), 778-784 (2003); the disclosures ofwhich are incorporated herein by reference. Again, there are concernsregarding conjugation site location and stoichiometry and the possibledisruption of antibody native conformation.

A number of methods are known for introducing free thiol groups intoantibodies without breaking native disulfide bonds, which methods can bepracticed with a ligand Z of this invention. Depending on the methodemployed, it may be possible to introduce a predictable number of freesulfhydryls at predetermined locations. In one approach, mutatedantibodies are prepared in which a cysteine is substituted for anotheramino acid. See, for example, Eigenbrot et al., U.S. Pat. No. 7,521,541B2 (2009); Chilkoti et al., Bioconjugate Chem. 1994, 5, 504-507;Urnovitz et al., U.S. Pat. No. 4,698,420 (1987); Stimmel et al., J.Biol. Chem., 275 (39), 30445-30450 (2000); Bam et al., U.S. Pat. No.7,311,902 B2 (2007); Kuan et al., J. Biol. Chem., 269 (10), 7610-7618(1994); Poon et al., J. Biol. Chem., 270 (15), 8571-8577 (1995). Inanother approach, an extra cysteine is added to the C-terminus See, e.g.Cumber et al., J. Immunol., 149, 120-126 (1992); King et al., CancerRes., 54, 6176-6185 (1994); Li et al., Bioconjugate Chem., 13, 985-995(2002); Yang et al., Protein Engineering, 16, 761-770 (2003); andOlafson et al., Protein Engineering Design & Selection, 17, 21-27(2004). A preferred method for introducing free cysteines is that taughtby Liu et al., WO 2009/026274 A1, in which a cysteine bearing amino acidsequence is added to the C-terminus of the heavy chain of an antibody.This method introduces a known number of cysteine residues (one perheavy chain) at a known location away from the antigen binding site. Thedisclosures of the documents cited in this paragraph are allincorporated herein by reference.

In yet another embodiment, lysine ε-amino groups can be modified withheterobifunctional reagents such as 2-iminothiolane orN-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), converting anε-amino group into a thiol or disulfide group—creating a cysteinesurrogate, as it were. However, this method suffers from the sameconjugation location and stoichiometry limitations associated withε-amino groups proper.

In yet another preferred embodiment, ligand Z is conjugated via thenucleophilic addition product of a thiol group to an acceptor moiety. Apreferred acceptor moiety is a maleimide group, whose reaction with anantibody thiol group is generically illustrated below. The thiol groupcan be a native one, or one introduced as described above.

Ligand Z can also be conjugated via a functional group adapted for usewith “click” chemistry, as discussed hereinbelow.Linker —(X^(D))_(a)C(X^(Z))_(b)—

As noted above, the linker portion of a conjugate of this inventioncomprises up to three elements: a cleavable group C and optional spacersX^(Z) and X^(D).

Cleavable group C is a group cleavable under physiological conditions,preferably selected such that it is relatively stable while theconjugate is in general circulation in the blood plasma, but is readilycleaved once the conjugate reaches its site of intended action, that is,near, at, or within the target cell. Preferably, the conjugate isinternalized by endocytosis by a target cell upon binding of antibody Zto an antigen displayed on the surface of the target cell. Subsequently,cleavage of group C occurs in a vesicular body of the target cell (anearly endosome, a late endosome, or, especially, a lysosome).

In one embodiment, group C is a pH sensitive group. The pH in bloodplasma is slightly above neutral, while the pH inside a lysosome isacidic, circa 5. Thus, a group C whose cleavage is acid catalyzed willcleave at a rate several orders of magnitude faster inside a lysosomethan in the blood plasma rate. Examples of suitable acid-sensitivegroups include cis-aconityl amides and hydrazones, as described in Shenet al., U.S. Pat. No. 4,631,190 (1986); Shen et al., U.S. Pat. No.5,144,011 (1992); Shen et al., Biochem. Biophys. Res. Commun. 102,1048-1054 (1981) and Yang et al., Proc. Natl Acad. Sci (USA), 85,1189-1193 (1988); the disclosures of which are incorporated herein byreference.

In another embodiment, group C is a disulfide. Disulfides can be cleavedby a thiol-disulfide exchange mechanism, at a rate dependent on theambient thiol concentration. As the intracellular concentration ofglutathione and other thiols is higher than their serum concentrations,the cleavage rate of a disulfide will be higher intracellularly.Further, the rate of thiol-disulfide exchange can be modulated byadjustment of the steric and electronic characteristics of the disulfide(e.g., an alkyl-aryl disulfide versus an alkyl-alkyl disulfide;substitution on the aryl ring, etc.), enabling the design of disulfidelinkages that have enhanced serum stability or a particular cleavagerate. For additional disclosures relating to disulfide cleavable groupsin conjugates, see, e.g., Thorpe et al., Cancer Res. 48, 6396-6403(1988); Santi et al., U.S. Pat. No. 7,541,530 B2 (2009); Ng et al., U.S.Pat. No. 6,989,452 B2 (2006); Ng et al., WO 2002/096910 A1; Boyd et al.,U.S. Pat. No. 7,691,962 B2; and Sufi et al., US 2010/0145036 A1; thedisclosures of which are incorporated herein by reference.

A preferred group C comprises a peptide bond that is cleaved,preferentially by a protease at the intended site of action, as opposedto by a protease in the serum. Typically, group C comprises from 1 to 20amino acids, preferably from 1 to 6 amino acids, more preferably from 1to 3 amino acids. The amino acid(s) can be natural and/or unnaturalα-amino acids. Natural amino acids are those encoded by the geneticcode, as well as amino acids derived therefrom, e.g., hydroxyproline,γ-carboxyglutamate, citrulline, and 0-phosphoserine. The term amino acidalso includes amino acid analogs and mimetics. Analogs are compoundshaving the same general H₂N(R)CHCO₂H structure of a natural amino acid,except that the R group is not one found among the natural amino acids.Examples of analogs include homoserine, norleucine,methionine-sulfoxide, and methionine methyl sulfonium. An amino acidmimetic is a compound that has a structure different from the generalchemical structure of an α-amino acid but functions in a manner similarto one. The term “unnatural amino acid” is intended to represent the “D”stereochemical form, the natural amino acids being of the “L” form.

Preferably, group C contains an amino acid sequence that is a cleavagerecognition sequence for a protease. Many cleavage recognition sequencesare known in the art. See, e.g., Matayoshi et al. Science 247: 954(1990); Dunn et al. Meth. Enzymol. 241: 254 (1994); Seidah et al. Meth.Enzymol. 244: 175 (1994); Thornberry, Meth. Enzymol. 244: 615 (1994);Weber et al. Meth. Enzymol. 244: 595 (1994); Smith et al. Meth. Enzymol.244: 412 (1994); and Bouvier et al. Meth. Enzymol. 248: 614 (1995); thedisclosures of which are incorporated herein by reference.

For conjugates that are not intended to be internalized by a cell, agroup C can be chosen such that it is cleaved by a protease present inthe extracellular matrix in the vicinity of the target tissue, e.g., aprotease released by nearby dying cells or a tumor-associated protease.Exemplary extracellular tumor-associated proteases are matrixmetalloproteases (MMP), thimet oligopeptidase (TOP) and CD10.

For conjugates that are designed to be internalized by a cell, group Cpreferably comprises an amino acid sequence selected for cleavage by anendosomal or lysosomal protease, especially the latter. Non-limitingexamples of such proteases include cathepsins B, C, D, H, L and S,especially cathepsin B. Cathepsin B preferentially cleaves peptides at asequence -AA²-AA¹- where AA¹ is a basic or strongly hydrogen bondingamino acid (such as lysine, arginine, or citrulline) and AA² is ahydrophobic amino acid (such as phenylalanine, valine, alanine, leucine,or isoleucine), for example Val-Cit (where Cit denotes citrulline) orVal-Lys. (Herein, amino acid sequences are written in the N-to-Cdirection, as in H₂N-AA²-AA¹-CO₂H, unless the context clearly indicatesotherwise.) For additional information regarding cathepsin-cleavablegroups, see Dubowchik et al., Biorg. Med. Chem. Lett. 8, 3341-3346(1998); Dubowchik et al., Bioorg. Med. Chem. Lett., 8 3347-3352 (1998);and Dubowchik et al., Bioconjugate Chem. 13, 855-869 (2002); thedisclosures of which are incorporated by reference. Another enzyme thatcan be utilized for cleaving peptidyl linkers is legumain, a lysosomalcysteine protease that preferentially cleaves at Ala-Ala-Asn.

In one embodiment, Group C is a peptide comprising the two-amino acidsequence -AA²-AA¹- wherein AA¹ is lysine, arginine, or citrulline andAA² is phenylalanine, valine, alanine, leucine or isoleucine. In anotherembodiment, C consists of a sequence of one to five amino acids,selected from the group consisting of Val-Cit, Ala-Val, Val-Ala-Val,Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Cit-Cit, Val-Lys, Ala-Ala-Asn, Lys,Cit, Ser, and Glu.

The preparation and design of cleavable groups C consisting of a singleamino acid is disclosed in Chen et al., US 2010/0113476 A1, thedisclosure of which is incorporated herein by reference.

Group C can also be a photocleavable one, for example a nitrobenzylether that is cleaved upon exposure to light.

Group C can be bonded directly to antibody Z or compound D; that is,spacers X^(Z) and X^(D), as the case may be, can be absent. For example,if group C is a disulfide, one of the two sulfurs can be a cysteineresidue or its surrogate on antibody Z. Or, group C can be a hydrazonebonded to an aldehyde on a carbohydrate side chain of the antibody. Or,group C can be a peptide bond formed with a lysine ε-amino group ofantibody Z. In a preferred embodiment, compound D is directly bonded togroup C via a peptidyl bond to a carboxyl or amine group in compound D.

When present, spacer X^(Z) provides spatial separation between group Cand antibody Z, lest the former sterically interfere with antigenbinding by latter or the latter sterically interfere with cleavage ofthe former. Further, spacer X^(Z) can be used to confer increasedsolubility or decreased aggregation properties to conjugates. A spacerX^(Z) can comprise one or more modular segments, which can be assembledin any number of combinations. Examples of suitable segments for aspacer X^(Z) are:

and combinations thereof, where the subscript q is 0 or 1 and thesubscript r is 1 to 24, preferably 2 to 4. These segments can becombined, such as shown below:

Spacer X^(D), if present, provides spatial separation between group Cand compound D, lest the latter interfere sterically or electronicallywith cleavage of the former. Spacer X^(D) also can serve to introduceadditional molecular mass and chemical functionality into a conjugate.Generally, the additional mass and functionality will affect the serumhalf-life and other properties of the conjugate. Thus, through judiciousselection of spacer groups, the serum half-live of a conjugate can bemodulated. Spacer X^(D) also can be assembled from modular segments, asdescribed above in the context of spacer X^(Z).

Spacers X^(Z) and/or X^(D), where present, preferably provide a linearseparation of from 4 to 25 atoms, more preferably from 4 to 20 atoms,between Z and C or D and C, respectively.

Either spacer X^(Z) or X^(D), or both, can comprise a self-immolatingmoiety. A self-immolating moiety is a moiety that (1) is bonded to groupC and either antibody Z or cytotoxin D and (2) has a structure such thatcleavage from group C initiates a reaction sequence resulting in theself-immolating moiety disbonding itself from antibody Z or cytotoxin D,as the case may be. In other words, reaction at a site distal fromantibody Z or cytotoxin D (cleavage from group C) causes the X^(Z)—Z orthe X^(D)-D bond to rupture as well. The presence of a self-immolatingmoiety is desirable in the case of spacer X^(D) because, if, aftercleavage of the conjugate, spacer X^(D) or a portion thereof were toremain attached to cytotoxin D, the biological activity of the lattermay be impaired. The use of a self-immolating moiety is especiallydesirable where cleavable group C is a polypeptide.

Exemplary self-immolating moieties (i)-(v) bonded to a hydroxyl or aminogroup on a partner molecule D are shown below:

The self-immolating moiety is the structure between dotted lines a andb, with adjacent structural features shown to provide context.Self-immolating moieties (i) and (v) are bonded to a compound D-NH₂(i.e., compound D is conjugated via an amino group), whileself-immolating moieties (ii), (iii), and (iv) are bonded to a compoundD-OH (i.e., compound D is conjugated via a hydroxyl or carboxyl group).Cleavage of the amide bond at dotted line b releases the amide nitrogenas an amine nitrogen, initiating a reaction sequence that results in thecleavage of the bond at dotted line a and the consequent release of D-OHor D-NH₂, as the case may be. For additional disclosures regardingself-immolating moieties, see Carl et al., J. Med. Chem., 24 (3),479-480 (1981); Carl et al., WO 81/01145 (1981); Dubowchik et al.,Pharmacology & Therapeutics, 83, 67-123 (1999); Firestone et al., U.S.Pat. No. 6,214,345 B1 (2001); Toki et al., J. Org. Chem. 67, 1866-1872(2002); Doronina et al., Nature Biotechnology 21 (7), 778-784 (2003)(erratum, p. 941); Boyd et al., U.S. Pat. No. 7,691,962 B2; Boyd et al.,US 2008/0279868 A1; Sufi et al., WO 2008/083312 A2; Feng, U.S. Pat. No.7,375,078 B2; and Senter et al., US 2003/0096743 A1; the disclosures ofwhich are incorporated by reference.

In another embodiment, an antibody targeting moiety and the cytotoxiccompound D are linked by a non-cleavable linker. Degradation of theantibody eventually reduces the linker to a small appended moiety thatdoes not interfere with the biological activity of cytotoxic compound D.

Compound D—Linker Compositions

Conjugates of this invention preferably are prepared by first joining acompound D and linker (X^(D))_(a)C(X^(Z))_(b) (where X^(D), C, X^(Z), a,and b are as defined for formula (II)) to form a drug-linker compositionrepresented by formula (III):D-(X^(D))_(a)C(X^(Z))_(b)—R³¹  (III)where R³¹ is a functional group suitable for reacting with a functionalgroup on antibody Z to form the conjugate. Examples of suitable groupsR³¹ include amino, azide, cyclooctyne,

where R³² is Cl, Br, F, mesylate, or tosylate and R³³ is Cl, Br, I, F,OH, —O—N-succinimidyl, —O-(4-nitrophenyl), —O-pentafluorophenyl, or—O-tetrafluorophenyl. Chemistry generally usable for the preparation ofsuitable moieties D-(X^(D))_(a)C(X^(Z))_(b)—R³¹ is disclosed in Ng etal., U.S. Pat. No. 7,087,600 B2 (2006); Ng et al., U.S. Pat. No.6,989,452 B2 (2006); Ng et al., U.S. Pat. No. 7,129,261 B2 (2006); Ng etal., WO 02/096910 A1; Boyd et al., U.S. Pat. No. 7,691,962 B2; Chen etal., U.S. Pat. No. 7,517,903 B2 (2009); Gangwar et al., U.S. Pat. No.7,714,016 B2 (2010); Boyd et al., US 2008/0279868 A1; Gangwar et al.,U.S. Pat. No. 7,847,105 B2 (2010); Gangwar et al., U.S. Pat. No.7,968,586 B2 (2011); Sufi et al., US 2010/0145036 A1; and Chen et al.,US 2010/0113476 A1; the disclosures of which are incorporated herein byreference.

Preferably reactive functional group —R³¹ is —NH₂, —OH, —CO₂H, —SH,maleimido, cyclooctyne, azido (—N₃), hydroxylamino (—ONH₂) orN-hydroxysuccinimido. Especially preferred functional groups —R³¹ areselected from the group consisting of:

An —OH group can be esterified with a carboxy group on the antibody, forexample, on an aspartic or glutamic acid side chain.

A —CO₂H group can be esterified with a —OH group or amidated with anamino group (for example on a lysine side chain) on the antibody.

An N-hydroxysuccinimide group is functionally an activated carboxylgroup and can conveniently be amidated by reaction with an amino group(e.g., from lysine).

A maleimide group can be conjugated with an —SH group on the antibody(e.g., from cysteine or from the chemical modification of the antibodyto introduce a sulfhydryl functionality), in a Michael additionreaction.

An —SH group is particularly useful for conjugation where the antibodyhas been modified to introduce a maleimide group thereto, in a Michaeladdition reaction that is the “mirror image” of that described above.Antibodies can be modified to have maleimide groups with N-succinimidyl4-(maleimidomethyl)-cyclohexanecarboxylate (SMCC) or its sulfonatedvariant sulfo-SMCC, both reagents being available from Sigma-Aldrich.

Azide and cyclooctyne are complementary functional groups that caneffect conjugation via so-called copper-free “click chemistry,” in whichthe azide adds across the strained alkyne bond of the cyclooctyne toform an 1,2,3-triazole ring. See, e.g., Agard et al., J. Amer. Chem.Soc. 2004, 126, 15046-15047; Best, Biochemistry 2009, 48, 6571-6584. Theazide can be the reactive functional group R³¹ in formula (III) and thecyclooctyne can be situated on the antibody or antigen binding portionthereof, or vice-versa. A cyclooctyne group can be provided by a DIBOreagent (available from Invitrogen/Molecular Probes, Eugene, Oreg.).

Techniques for introducing non-natural amino acids into antibodies canbe utilized, with the non-natural amino acid providing a functionalityfor conjugation with the reactive functional group. For instance, thenon-natural amino acid p-acetylphenylalanine can be incorporated into anantibody or other polypeptide, as taught in Tian et al., WO 2008/030612A2 (2008). The ketone group in p-acetylphenyalanine can be a conjugationsite by the formation of an oxime with a hydroxylamino reactivefunctional group. Alternatively, the non-natural amino acidp-azidophenylalanine can be incorporated into an antibody to provide anazide functional group for conjugation via click chemistry. Non-naturalamino acids can also be incorporated into an antibody or otherpolypeptide using cell-free methods, as taught in Goerke et al., US2010/0093024 A1 (2010) and Goerke et al., Biotechnol. Bioeng. 2009, 102(2), 400-416.

An amine (NH₂) group can be used for conjugation using the enzymetransglutaminase, as taught in Jeger et al., Angew. Chem. Int. Ed. 2010,49, 9995-9997.

Conjugation can also be effected using the enzyme Sortase A, as taughtin Levary et al., PLoS One 2011, 6(4), e18342; Proft, Biotechnol. Lett.2010, 32, 1-10; Ploegh et al., WO 2010/087994 A2 (2010); and Mao et al.,WO 2005/051976 A2 (2005). The Sortase A recognition motif (typicallyLPXTG, where X is any natural amino acid) may be located on the ligand Zand the nucleophilic acceptor motif (typically GGG) may be the group R³¹in formula (III), or vice-versa.

The group D in the formulae [D(X^(D))_(a)C(X^(Z))_(b)]_(m)Z andD-(X^(D))_(a)C(X^(Z))_(b)—R³¹ preferably has a structure according toformula (D-a)

or formula (D-b)

wherein Y is H or NO₂; R^(4a) is H, Me, or Et; R^(3a) and R^(3b) areindependently H, Me, or Et; and R⁶ is C₁-C₅ alkyl, CH₂OC(═O)C₁-C₅ alkyl,or (CH₂)₁₋₂C₆H₅.

Examples of such groups D include:

wherein R⁷ is H, Me, or Et.

Examples of compositions according to formulaD-(X^(D))_(a)C(X^(Z))_(b)—R³¹ include the ones shown immediatelyfollowing; along with their pharmaceutically acceptable salts:

A preferred drug-linker compound has a structure represented by formula(III-a):

where

-   -   R^(3a) and R^(3b) are independently H, Me, or Et;    -   R⁶ is Me, Et, or n-Pr;    -   AA^(a) and each AA^(b) are independently selected from the group        consisting of alanine, β-alanine, γ-aminobutyric acid, arginine,        asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline,        cysteine, glutamic acid, glutamine, glycine, histidine,        isoleucine, leucine, lysine, methionine, norleucine, norvaline,        ornithine, phenylalanine, proline, serine, threonine,        tryptophan, tyrosine, and valine;    -   p is 1, 2, 3, or 4;    -   q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 2, 3, or 4);    -   r is 1, 2, 3, 4, or 5;    -   s is 0 or 1; and    -   R³¹ is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.

In formula (III-a), -AA^(a)-[AA^(b)]_(p)- represents a polypeptide whoselength is determined by the value of p (e.g., dipeptide if p is 1,tetrapeptide if p is 3, etc.). AA^(a) is at the carboxy terminus of thepolypeptide and its carboxyl group forms a peptide (amide) bond with theanilino nitrogen of the drug. Conversely, the last AA^(b) is at theamino terminus of the polypeptide and its alpha-amino group forms apeptide bond with

if s is 1 and with

if s is 0.

A more preferred drug-linker compound has a structure represented byformula (III-b):

where

R⁶ is Me or n-Pr;

q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (preferably 2, 3, or 4);

r is 1, 2, 3, 4, or 5;

s is 0 or 1; and

R³¹ is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.Preparation of Conjugates

The following is an illustrative procedure, based on introduction offree thiol groups into an antibody by reaction of lysine ε-amino groupswith 2-iminothiolane, followed by reaction with a maleimide-containingdrug-linker moiety such as described above. Initially the antibody isbuffer exchanged into 0.1 M phosphate buffer (pH 8.0) containing 50 mMNaCl and 2 mM diethylene triamine pentaacetic acid (DTPA) andconcentrated to 5-10 mg/mL. Thiolation is achieved through addition of2-iminothiolane to the antibody. The amount of 2-iminothiolane to beadded can be determined by a preliminary experiment and varies fromantibody to antibody. In the preliminary experiment, a titration ofincreasing amounts of 2-iminothiolane is added to the antibody, andfollowing incubation with the antibody for 1 h at RT (room temperature,circa 25° C.), the antibody is desalted into 50 mM pH 6.0 HEPES bufferusing a SEPHADEX™ G-25 column and the number of thiol groups introduceddetermined rapidly by reaction with dithiodipyridine (DTDP). Reaction ofthiol groups with DTDP results in liberation of thiopyridine, which canbe monitored spectroscopically at 324 nm. Samples at a proteinconcentration of 0.5-1.0 mg/mL are typically used. The absorbance at 280nm can be used to accurately determine the concentration of protein inthe samples, and then an aliquot of each sample (0.9 mL) is incubatedwith 0.1 mL DTDP (5 mM stock solution in ethanol) for 10 min at RT.Blank samples of buffer alone plus DTDP are also incubated alongside.After 10 min, absorbance at 324 nm is measured and the number of thiolgroups is quantitated using an extinction coefficient for thiopyridineof 19,800 M⁻¹.

Typically a thiolation level of about three thiol groups per antibody isdesirable. For example, with some antibodies this can be achieved byadding a 15-fold molar excess of 2-iminothiolane followed by incubationat RT for 1 h. The antibody is then incubated with 2-iminothiolane atthe desired molar ratio and then desalted into conjugation buffer (50 mMpH 6.0 HEPES buffer containing 5 mM glycine and 2 mM DTPA). Thethiolated material is maintained on ice while the number of thiolsintroduced is quantitated as described above.

After verification of the number of thiols introduced, the drug-linkermoiety is added at a 3-fold molar excess per thiol. The conjugationreaction is allowed to proceed in conjugation buffer also containing afinal concentration of 5% dimethylsulfoxide (DMSO), or similaralternative solvent. Commonly, the drug-linker stock solution isdissolved in 100% DMSO. The stock solution is added directly to thethiolated antibody, which has enough DMSO added to bring the finalconcentration to 10%, or pre-diluted in conjugation buffer containing afinal concentration of 10% DMSO, followed by addition to an equal volumeof thiolated antibody.

The conjugation reaction mixture is incubated at RT for 2 h withstirring. Following incubation, the conjugation reaction mixture iscentrifuged and filtered through a 0.2 μm filter. Purification of theconjugate can be achieved through chromatography using a number ofmethods. In one method, the conjugate is purified using size-exclusionchromatography on a SEPHACRYL™ S200 column pre-equilibrated with 50 mMpH 7.2 HEPES buffer containing 5 mM glycine and 150 mM NaCl.Chromatography is carried out at a linear flow rate of 28 cm/h.Fractions containing conjugate are collected, pooled and concentrated.In an alternative method, purification can be achieved throughion-exchange chromatography. Conditions vary from antibody to antibodyand should to be optimized in each case. For example, antibody-drugconjugate reaction mix is applied to an SP-SEPHAROSE™ columnpre-equilibrated in 50 mM pH 5.5 HEPES containing 5 mM glycine. Theantibody conjugate is eluted using a gradient of 0-1 M NaCl inequilibration buffer at pH 5.5. Relevant fractions containing theconjugate are pooled and dialyzed against formulation buffer (50 mM pH7.2 HEPES buffer containing 5 mM glycine and 100 mM NaCl).

Those skilled in the art will understand that the above-describedconditions and methodology are exemplary and non-limiting and that otherapproaches for conjugation are known in the art and usable in thepresent invention.

A conjugate prepared by the procedure described above is represented byformula (II-1). It is a conjugate of compound (III-1) and theanti-mesothelin antibody 6A4 (Terrett et al. 2012):

Those skilled in the art will appreciate that such a conjugatepreparation may have moieties with different substitution ratios,typically ranging from 1 to 5, and that such preparation can berepresented by formula (II-1′):

where R⁶ is Me or n-Pr and Ab is an antibody. The antibody preferably isan anti-CD70, anti-mesothelin, or anti-glypican-3 antibody.Pharmaceutical Compositions

In another aspect, the present disclosure provides a pharmaceuticalcomposition comprising a compound of the present invention, or of aconjugate thereof, formulated together with a pharmaceuticallyacceptable carrier or excipient. It may optionally contain one or moreadditional pharmaceutically active ingredients, such as an antibody oranother drug. The pharmaceutical compositions can be administered in acombination therapy with another therapeutic agent, especially anotheranti-cancer agent.

The pharmaceutical composition may comprise one or more excipients.Excipients that may be used include carriers, surface active agents,thickening or emulsifying agents, solid binders, dispersion orsuspension aids, solubilizers, colorants, flavoring agents, coatings,disintegrating agents, lubricants, sweeteners, preservatives, isotonicagents, and combinations thereof. The selection and use of suitableexcipients is taught in Gennaro, ed., Remington: The Science andPractice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), thedisclosure of which is incorporated herein by reference.

Preferably, a pharmaceutical composition is suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the active compound may be coated in a material toprotect it from the action of acids and other natural conditions thatmay inactivate it. The phrase “parenteral administration” means modes ofadministration other than enteral and topical administration, usually byinjection, and includes, without limitation, intravenous, intramuscular,intraarterial, intrathecal, intracapsular, intraorbital, intracardiac,intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. Alternatively, the pharmaceuticalcomposition can be administered via a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually or topically.

Pharmaceutical compositions can be in the form of sterile aqueoussolutions or dispersions. They can also be formulated in amicroemulsion, liposome, or other ordered structure suitable to achievehigh drug concentration. The compositions can also be provided in theform of lyophilates, for reconstitution in water prior toadministration.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated and the particular mode of administration and willgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of one hundred per cent, this amount will rangefrom about 0.01 per cent to about ninety-nine percent of activeingredient, preferably from about 0.1 per cent to about 70 per cent,most preferably from about 1 per cent to about 30 per cent of activeingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide a therapeutic response. Forexample, a single bolus may be administered, several divided doses maybe administered over time, or the dose may be proportionally reduced orincreased as indicated by the exigencies of the situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. “Dosageunit form” refers to physically discrete units suited as unitary dosagesfor the subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic response, in association with the required pharmaceuticalcarrier.

The dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01to 5 mg/kg, of the host body weight. For example dosages can be 0.3mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kgbody weight or 10 mg/kg body weight or within the range of 1-10 mg/kg.Exemplary treatment regimens are administration once per week, onceevery two weeks, once every three weeks, once every four weeks, once amonth, once every 3 months, or once every three to 6 months. Preferreddosage regimens include 1 mg/kg body weight or 3 mg/kg body weight viaintravenous administration, using one of the following dosing schedules:(i) every four weeks for six dosages, then every three months; (ii)every three weeks; (iii) 3 mg/kg body weight once followed by 1 mg/kgbody weight every three weeks. In some methods, dosage is adjusted toachieve a plasma antibody concentration of about 1-1000 μg/mL and insome methods about 25-300 μg/mL.

A “therapeutically effective amount” of a compound of the inventionpreferably results in a decrease in severity of disease symptoms, anincrease in frequency and duration of disease symptom-free periods, or aprevention of impairment or disability due to the disease affliction.For example, for the treatment of tumor-bearing subjects, a“therapeutically effective amount” preferably inhibits tumor growth byat least about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. A therapeutically effectiveamount of a therapeutic compound can decrease tumor size, or otherwiseameliorate symptoms in a subject, which is typically a human but can beanother mammal.

The pharmaceutical composition can be a controlled or sustained releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See,e.g., Sustained and Controlled Release Drug Delivery Systems, J. R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Therapeutic compositions can be administered via medical devices such as(1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and4,596,556); (2) micro-infusion pumps (U.S. Pat. No. 4,487,603); (3)transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion apparati(U.S. Pat. Nos. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S.Pat. Nos. 4,439,196 and 4,475,196); the disclosures of which areincorporated herein by reference.

In certain embodiments, the pharmaceutical composition can be formulatedto ensure proper distribution in vivo. For example, to ensure that thetherapeutic compounds of the invention cross the blood-brain barrier,they can be formulated in liposomes, which may additionally comprisetargeting moieties to enhance selective transport to specific cells ororgans. See, e.g. U.S. Pat. Nos. 4,522,811; 5,374,548; 5,416,016; and5,399,331; V. V. Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa etal., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al.(1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. AgentsChemother. 39:180; Briscoe et al. (1995) Am. J. Physiol. 1233:134;Schreier et al. (1994) J. Biol. Chem. 269:9090; Keinanen and Laukkanen(1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods4:273.

Uses

Compounds of this invention or their conjugates can be used for treatingdiseases such as, but not limited to, hyperproliferative diseases,including: cancers of the head and neck which include tumors of thehead, neck, nasal cavity, paranasal sinuses, nasopharynx, oral cavity,oropharynx, larynx, hypopharynx, salivary glands, and paragangliomas;cancers of the liver and biliary tree, particularly hepatocellularcarcinoma; intestinal cancers, particularly colorectal cancer; ovariancancer; small cell and non-small cell lung cancer (SCLC and NSCLC);breast cancer sarcomas, such as fibrosarcoma, malignant fibroushistiocytoma, embryonal rhabdomyosarcoma, leiomysosarcoma,neurofibrosarcoma, osteosarcoma, synovial sarcoma, liposarcoma, andalveolar soft part sarcoma; leukemias such as acute promyelocyticleukemia (APL), acute myelogenous leukemia (AML), acute lymphoblasticleukemia (ALL), and chronic myelogenous leukemia (CML); neoplasms of thecentral nervous systems, particularly brain cancer; multiple myeloma(MM), lymphomas such as Hodgkin's lymphoma, lymphoplasmacytoid lymphoma,follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, mantlecell lymphoma, B-lineage large cell lymphoma, Burkitt's lymphoma, andT-cell anaplastic large cell lymphoma. Clinically, practice of themethods and use of compositions described herein will result in areduction in the size or number of the cancerous growth and/or areduction in associated symptoms (where applicable). Pathologically,practice of the method and use of compositions described herein willproduce a pathologically relevant response, such as: inhibition ofcancer cell proliferation, reduction in the size of the cancer or tumor,prevention of further metastasis, and inhibition of tumor angiogenesis.The method of treating such diseases comprises administering atherapeutically effective amount of an inventive combination to asubject. The method may be repeated as necessary. Especially, the cancercan be renal, lung, gastric, or ovarian cancer.

Compounds of this invention or their conjugates can be administered incombination with other therapeutic agents, including antibodies,alkylating agents, angiogenesis inhibitors, antimetabolites, DNAcleavers, DNA crosslinkers, DNA intercalators, DNA minor groove binders,enediynes, heat shock protein 90 inhibitors, histone deacetylaseinhibitors, immunomodulators, microtubule stabilizers, nucleoside(purine or pyrimidine) analogs, nuclear export inhibitors, proteasomeinhibitors, topoisomerase (I or II) inhibitors, tyrosine kinaseinhibitors, and serine/threonine kinase inhibitors. Specific therapeuticagents include adalimumab, ansamitocin P3, auristatin, bendamustine,bevacizumab, bicalutamide, bleomycin, bortezomib, busulfan, callistatinA, camptothecin, capecitabine, carboplatin, carmustine, cetuximab,cisplatin, cladribin, cytarabin, cryptophycins, dacarbazine, dasatinib,daunorubicin, docetaxel, doxorubicin, duocarmycin, dynemycin A,epothilones, etoposide, floxuridine, fludarabine, 5-fluorouracil,gefitinib, gemcitabine, ipilimumab, hydroxyurea, imatinib, infliximab,interferons, interleukins, β-lapachone, lenalidomide, irinotecan,maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate,mitomycin C, nilotinib, oxaliplatin, paclitaxel, procarbazine,suberoylanilide hydroxamic acid (SAHA), 6-thioguanidine, thiotepa,teniposide, topotecan, trastuzumab, trichostatin A, vinblastine,vincristine, and vindesine.

EXAMPLES

The practice of this invention can be further understood by reference tothe following examples, which are provided by way of illustration andnot of limitation.

Example 1 Compound (III-1)

This example describes the synthesis of compound (III-1), thecorresponding scheme being shown in combined FIGS. 1, 2 a-2 b, and 3.

Compound 2. A mixture of compound 1 (6 g, 16.6 mmol; prepared accordingto Peltier et al. 2006) and paraformaldehyde (9.94 g, 331 mmol) intoluene (150 mL) was heated in a sealed vessel at 70° C. for 24 h. Thinlayer chromatography (TLC) showed that the reaction was complete. Thereaction mixture was filtered through CELITE™ filter media and thefilter cake was washed thoroughly with toluene. After evaporation of thesolvent, the crude product was purified by flash chromatography elutingfrom silica gel with a gradient of 0-70% ethyl acetate (EtOAc) indichloromethane (DCM) to afford 4.76 g of compound 2 as a light yellowoil. MS: (+) m/z 375.2 (M+1).

Compound 3. Hydrochloric acid (4.0 M in 1,4-dioxane, 12.24 mL, 50.8mmol) was added drop-wise to a solution of compound 2 (4.76 g, 12.7mmol) in acetonitrile (62 mL) and methanol (6.8 mL), in the presence ofsupport-bound cyanoborohydride (MP-BH₃CN) resin (4.85 g, 12.7 mmol). Thereaction mixture was stirred at room temperature (RT) for 3 h. LCMSshowed the reaction went to completion. The resin was filtered off andwashed with acetonitrile-methanol mixture. After evaporation of thesolvent, the crude product was purified by flash chromatography elutingfrom silica gel with a gradient of 0-10% methanol in DCM containing 1%NH₄OH to afford crude compound 3.

The product fractions were concentrated, diluted with EtOAc, and washedonce with saturated aq. NaHCO₃ to remove excess ammonium salts. Theaqueous fraction was back-extracted once with EtOAc. The combinedorganic phases were dried and concentrated to afford 2.82 g of compound3 as a foamy solid. MS: (+) m/z 273.2 (M+1).

Compound 4. Polymer-bound N-benzyl-N-cyclohexylcarbodiimide (Aldrich,4.5 g, 5.21 mmol) was added to a solution of compound 3 (1.42 g, 5.21mmol), t-butanol (0.72 g, 5.32 mmol) and Boc-protected isoleucine 3a(1.27 g, 5.47 mmol) in DCM (48 mL) at 0° C. The reaction mixture wasstirred at RT overnight. The resin was filtered off and washed with DCM.The filtrate was concentrated, diluted with EtOAc, and washed once withsaturated aq. NaHCO₃. The aqueous solution was extracted twice withEtOAc. The combined organic layers were dried, filtered andconcentrated. The crude product was purified by flash chromatographyeluting from silica gel with a gradient of 0-10% methanol in DCMcontaining 1% NH₄OH to afford fractons containing intermediate product3b.

The product-containing fractions were combined, concentrated, dilutedwith EtOAc, and washed with saturated aq. NaHCO₃ to remove excessammonium salts. The aqueous fraction was back-extracted once with EtOAc.The combined organic phases were dried and concentrated to affordintermediate product 3b as a white solid.

Intermediate product 3b in toluene (50 mL) was heated to 90° C. in asealed vessel overnight, with stirring. LCMS showed the reaction went tocompletion. The solvent was evaporated. The crude product was purifiedby flash chromatography eluting from silica gel with a gradient of0-100% EtOAc in hexanes to afford 1.3 g of compound 4 as a light yellowsolid. MS: (+) m/z 486.3 (M+1).

Compound 5. Trifluoroacetic acid (TFA, 26 mL) was added to a mixture ofcompound 4 in DCM (26 mL). After stirring at RT for 30 min, LCMS showedthe reaction was complete. The solution was concentrated, diluted withEtOAC, and washed once with saturated aq. NaHCO₃. The aqueous solutionwas back-extracted twice with EtOAc. The combined organic layers weredried, filtered, and concentrated to afford 1.03 g of compound 5 aswhite solid. MS: (+) m/z 386.3 (M+1).

Compound 6. DCC (0.664 g, 3.22 mmol) was added to a mixture of compound5 (1.03 g, 2.68 mmol), (R)-1-methylpiperidine-2-carboxylic acid 5a (0.4g, 2.81 mmol; prepared according to Peltier et al. 2006), and t-butanol(0.369 g, 2.73 mmol) in DCM at 0° C. The reaction mixture was allowed towarm to RT and stirred at RT overnight. The solid was filtered off, andthe filtrate was concentrated. The residue was dissolved in EtOAc andwashed once with saturated aq. NaHCO₃. The aqueous solution wasback-extracted twice with EtOAc. The combined organic layers were dried,filtered, and concentrated. The crude product was purified by flashchromatography eluting from silica gel with a gradient of 0-20% methanolin DCM to afford 1.23 g of compound 6 as a light yellow solid. MS: (+)m/z 511.4 (M+1).

Compound 7. A mixture of N,N-diisopropylethylamine (DIEA, also referredto as DIPEA, 0.972 mL, 5.58 mmol), bis(4-nitrophenyl) carbonate (BNPC,1.698 g, 5.58 mmol) and compound 6 (0.57 g, 1.116 mmol) inN,N-dimethylformamide (DMF, 10 mL) was stirred at RT overnight. LCMSshowed the reaction went to completion. The solvent was evaporated. Thecrude product was purified by silica gel flash chromatography with agradient of 0-20% methanol in DCM to afford 0.68 g of compound 7 as ayellow oil. MS: (+) m/z 676.4 (M+1).

Compound 8. Methylamine in methanol (2.0 M, 0.089 mL, 0.178 mmol) wasadded to compound 7 (0.1 g, 0.148 mmol) in methanol (1 mL). After thereaction mixture was stirred at RT for 10 min, LCMS showed the reactionwas complete. The solvent was evaporated to afford 0.084 g of compound8. MS: (+) m/z 568.4 (M+1).

Compound 9. Lithium hydroxide (7.09 mg, 0.296 mmol) in water (0.5 mL)was added to a solution of compound 8 (0.084 g, 0.148 mmol) in1,4-dioxane (0.5 mL) at RT. After the reaction mixture was stirred at RTfor 2 h, LCMS showed the reaction was complete. The solvent wasevaporated. The crude product was purified by flash chromatographyeluting from silica gel with a gradient of 0-30% methanol in DCM toafford 0.075 g of compound 9 as a white solid. MS: (+) m/z 554.4 (M+1).

Compound 10. Triethylamine (11.73 mL, 84 mmol) was added to a mixture ofdi-t-butyldicarbonate (BOC₂O, 10.57 mL, 46.0 mmol) and (5)-methyl2-amino-3-(4-nitrophenyl)propanoate hydrochloride 9a (10 g, 38.4 mmol)in acetonitrile (300 mL) at 0° C. The reaction mixture was allowed towarm to RT, and stirred at RT overnight. LCMS showed the reaction wentto completion. The reaction mixture was concentrated, and the productwas re-dissolved in 200 mL of diethyl ether. The solid was filtered off,and the filtrate was concentrated. The crude product was purified byflash chromatography eluting from silica gel with a gradient of 0-50%EtOAc in hexanes to afford 11.3 g of a Boc protected intermediate as awhite solid.

Pd/C catalyst (10 wt. %, 0.85 g, 7.99 mmol) was added to a solution ofthe Boc protected intermediate (15 g, 46.2 mmol) in MeOH (200 mL). Thereaction mixture was stirred under a hydrogen atmosphere overnight. ThePd/C catalyst was filtered, and the filtrate was concentrated to afford13.6 g of compound 10 as a white solid. MS: (+) m/z 195.2 (M+1-Boc).

Compound 11. Pyridine (5.77 mL, 71.3 mmol) was added to a solution ofbenzyl chloroformate (10.18 mL, 71.3 mmol) and compound 10 (17.5 g, 59.5mmol) in DCM (185 mL) at 0° C. The reaction mixture was allowed to warmto RT and was stirred at RT overnight. The reaction was quenched byaddition of saturated aq. NaHCO₃, and washed with brine. The organiclayer was dried, filtered, and concentrated. The crude product waspurified by flash chromatography eluting from silica gel with a gradientof 0-50% EtOAc in hexanes to afford 22.6 g of compound 11 as a colorlessoil. MS: (+) m/z 329.2 (M+1-Boc).

Compound 12. Diisobutylaluminum hydride (DIBAL-H) in hexanes (1M, 26.5mL, 26.5 mmol) was added to a solution of compound 11 (5.17 g, 12.07mmol) in DCM (39 mL) at −78° C. The reaction mixture was stirred at −78°C. for 2 h. Acetic acid (24 mL) and toluene (36 mL) were added at −78°C. The reaction mixture was warmed to RT. Tartaric acid (10% aq., 69 mL)was added to the reaction mixture. The aqueous solution was extractedwith hexanes and EtOAc (v/v 1:1) mixture. The combined organic layerswere dried, filtered, and concentrated. The crude product was purifiedby flash chromatography eluting from silica gel with a gradient of 0-50%EtOAc in hexanes to afford 3.12 g of compound 12 as a white solid. MS:(+) m/z 299.2 (M+1-Boc).

Compound 13. Dibutyl(((trifluoromethyl)sulfonyl)oxy)borane (Bu₂BOTf, 1Min DCM, 8.61 mL, 8.61 mmol) and DIEA (1.637 mL, 9.40 mmol) were added toa solution of (S)-4-isopropyl-3-propionyloxazolidin-2-one 12a (1.450 g,7.83 mmol) in DCM (7.8 mL) at 0° C. The reaction mixture was stirred at0° C. for 45 min. A solution of compound 12 (3.12 g, 7.83 mmol) in DCM(7.8 mL) was added to the reaction mixture at −78° C. The reactionmixture was allowed to warm up to RT overnight. Sodium phosphate buffer(pH 7, 29 mL) was added. The aqueous solution was extracted with DCM.The combined organic layers were washed with brine, dried, filtered, andconcentrated.

The residue was re-dissolved in methanol (130 mL) and cooled to 0° C.Aqueous H₂O₂ (30%, 39.7 mL) was added to the reaction mixture at 0° C.The reaction mixture was stirred at 0° C. for 4 h. Water (39 mL) wasadded. Some of the solvent (MeOH) was evaporated. The aqueous solutionwas extracted with EtOAc. The combined organic layers were washed with5% NaHCO₃ solution and brine, dried, filtered, and concentrated. Thecrude product was purified by flash chromatography eluting from silicagel with a gradient of 0-50% EtOAc in hexanes to afford 4.23 g ofcompound 13 as a colorless oil. MS: (+) m/z 484.3 (M+1-Boc).

Compound 14. Di(1H-imidazol-1-yl)methanethione (1.5 g, 8.42 mmol) wasadded to a solution of compound 13 (2.46 g, 4.21 mmol) in THF (20 mL).The reaction mixture was refluxed overnight. LCMS showed the reactionwent to completion. The solvent was evaporated. The crude product waspurified by silica gel flash chromatography with a gradient of 0-50%EtOAc in hexanes to afford 1.25 g of compound 14 as a white solid. MS:(+) m/z 694.3 (M+1).

Compound 15. (E)-2,2′-(diazene-1,2-diyl)bis(2-methylpropanenitrile)(AIBN, 0.016 g, 0.095 mmol) was added to a solution of compound 14 (1.78g, 2.57 mmol) and tributylstannane (Bu₃SnH, 1.380 mL, 5.13 mmol). Thereaction mixture was refluxed for 30 min (oil bath temperature at 142°C.). The solvent was evaporated. The crude product was purified by flashchromatography eluting from silica gel with a gradient of 0-33% EtOAc inhexanes to afford 0.84 g of compound 15 as a light yellow oil. MS: (+)m/z 468.3 (M+1-Boc).

Compound 16. LiOH (0.071 g, 2.96 mmol) in water (3.7 mL) was added to asolution of compound 15 (0.84 g, 1.480 mmol) in tetrahydrofuran (THF,11.4 mL), followed by addition of 30% aq. H₂O₂ (0.271 mL, 8.88 mmol) at0° C. After the reaction mixture was stirred at 0° C. for 4 h, 20 mL of1.33 M aq. Na₂SO₃ was added to quench the reaction. Hydrochloric acid (1M) was added to adjust the pH to 2-3. The resulting aqueous solution wasextracted with DCM. The combined organic layers were dried, filtered,and concentrated. The crude product was purified by flash chromatographyeluting from silica gel with a gradient of 0-75% EtOAc in hexanes toafford 0.53 g of compound 16 as a colorless oil. MS: (+) m/z 357.3(M+1-Boc).

Compound 17. Concentrated hydrochloric acid (4 drops) was added to asolution of 2,2-dimethoxypropane (3.53 mL, 28.7 mmol) and compound 16(0.53 g, 1.161 mmol) in methanol (17.7 mL). The reaction mixture wasstirred at RT overnight. LCMS showed the reaction went to completion.LCMS also showed the formation of some deprotected byproduct 16a. Thesolvent was evaporated.

Triethylamine (2.2 eq., 0.36 mL) was added to a solution of the aboveresidue and BOC₂O (1.2 eq., 304.3 mg) in acetonitrile at RT, tore-protect by-product 16a. The reaction mixture was stirred at RT for 2h. LCMS showed the reaction went to completion. The solvent wasevaporated. Water (7 mL) was added, and the aqueous solution wasextracted with EtOAc. The combined organic layers were dried, filtered,and concentrated. The crude product was purified by flash chromatographyeluting from silica gel with a gradient of 0-50% EtOAc in hexanes toafford 0.3 g of compound 17 as a colorless oil. MS: (+) m/z 371.3(M+1-Boc).

Compound 18. A mixture of compound 17 (0.223 g, 0.474 mmol) and Pd/C 10wt % (20 mg, 0.474 mmol) in methanol (6 mL) was stirred under H₂overnight. The Pd/C catalyst was filtered off, and the filtrateconcentrated. The crude product was purified by flash chromatographyeluting from silica gel with a gradient of 0-50% EtOAc in hexanes toafford 0.112 g of compound 18 as a white solid. MS: (+) m/z 237.2(M+1-Boc).

Compound 19. A mixture of compound 18 (0.204 g, 0.606 mmol),N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.174g, 0.910 mmol), and Fmoc-protected citrulline 18a (0.361 g, 0.910 mmol)in DMF (12.4 mL) was stirred at RT overnight. Saturated NH₄Cl solution(20 mL) was added to quench the reaction. The aqueous solution wasextracted with EtOAc. The combined organic layers were dried, filtered,and concentrated. The crude product was purified by silica gel flashchromatography with a gradient of 0-30% MeOH in DCM to afford 0.25 g ofcompound 19 as a white solid. MS: (+) m/z 716.4 (M+1).

Compound 20. Piperidine (0.5 mL, 5.06 mmol) was added to a solution ofcompound 19 (0.25 g, 0.349 mmol) in DMF (5 mL). After the reactionmixture was stirred at RT for 20 min, the solvent was evaporated toafford the Fmoc-deprotected intermediate as a residue.

DIEA was added to a solution of(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)-amino)-3-methylbutanoic acid19a (0.142 g, 0.418 mmol) andN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (HATU, 0.146 g, 0.383 mmol) in DMF (2 mL), adjustingthe pH to 8-9. After the reaction mixture was stirred at RT for 5 min,the above residue in DMF (1 mL) and DIEA were added to the reactionmixture, adjusting the pH to 8-9. After the reaction mixture was stirredat RT for 15 min, 20 mL of water containing 8 mL of 0.1% TFA water wasadded. The aqueous solution was extracted with EtOAc. The combinedorganic layers were dried, filtered, and concentrated. The crude productwas purified by flash chromatography eluting from silica gel with agradient of 0-20% MeOH in DCM to afford 0.24 g of compound 20 as a whitesolid. MS: (+) m/z 815.4 (M+1).

Compound 21. Piperidine (0.3 mL) was added to a solution of compound 20in DMF (3 mL). The reaction mixture was stirred at RT for 1 h. LCMSshowed the reaction went to completion. The solvent was evaporated.

Lithium hydroxide (0.028 g, 1.176 mmol) in water (2 mL) was added to asolution of the above residue in THF (4 mL). After the reaction mixturewas stirred at RT for 4 h, aq. HCl (0.1N) was added to acidify thereaction mixture (pH 2-3). The solvent was partially evaporated, andlyophilized to afford compound 21 as a white solid. MS: (+) m/z 579.4(M+1).

Compound 22. DIEA was added to a mixture of ε-maleimidocaproic acidN-hydroxysuccinimide ester 21a (Tokyo Chemical Industry, 64.7 mg, 0.210mmol) and compound 21 (81 mg, 0.14 mmol) in DMF (3 mL), adjusting pH8-9. After the reaction mixture was stirred at RT for 2 h, 10 mL of 1:1(v/v) mixture of acetonitrile and water containing 0.1% TFA was added.The product 22 was purified by preparative high performance liquidchromatography (HPLC). MS: (+) m/z 772.5 (M+1).

Compound 23. 2,2,2-Trifluoroacetic acid (0.7 mL, 0.013 mmol) was addedto a mixture of compound 22 (30 mg, 0.039 mmol) in DCM (1 mL) at RT.After the reaction mixture was stirred at RT for 10 min, LCMS showed thereaction went to completion. The solvent was evaporated, affordingcompound 23. MS: (+) m/z 672.4 (M+1).

Compound (III-1). DIEA was added to a solution of compound 9 (23.66 mg,0.043 mmol) and HATU (14.77 mg, 0.039 mmol) in DMF (1 mL). The pH of thereaction mixture was adjusted to 8-9. After the reaction mixture wasstirred at RT for 10 min, compound 23 (26.1 mg, 0.039 mmol) in DMF (1mL) and DIEA were added. The pH of the reaction solution was adjusted to8-9. After the reaction mixture was stirred at RT for 10 min, LCMSshowed the reaction was complete. The reaction was quenched by additionof 10 mL 1:1 (v/v) mixture of water containing 0.1% TFA andacetonitrile. The product compound (III-1) was purified by prep HPLC.MS: (+) m/z 1207.7 (M+1).

Compounds such as (III-1), having a maleimido group, can be used toprepare conjugates by reaction with a sulfhydryl group on an antibody orother ligand. The sulfhydryl group can be one from a cysteine residue orone obtained by derivatization of a lysine residue with 2-iminothiolane.

Example 2 Compound (III-2)

This example describes a synthesis of compound (III-2), thecorresponding scheme being shown in FIG. 4.

Compound 24. N-Ethyl-N-isopropylpropan-2-amine (0.556 mL, 3.19 mmol) wasadded to a solution of glycine tert-butyl ester hydrochloride 23a (0.209g, 1.596 mmol), Fmoc-aminoxyacetic acid 23b (0.5 g, 1.596 mmol) and HATU(0.607 g, 1.596 mmol) in DMF (5 mL) at RT. After the reaction mixturewas stirred at RT for 1 h, 0.1% aq. TFA (20 mL) was added. The aqueoussolution was extracted with EtOAc, and the combined organic layers weredried, filtered, and concentrated. The crude product was purified byflash chromatography eluting from silica gel with a gradient of 0-70%EtOAc in hexanes to afford 0.45 g of compound 24 as a colorless oil. MS:(+) m/z 449.2 (M+23).

Compound 25. TFA (3 mL, 1.437 mmol) was added to a solution of compound24 (0.45 g, 1.055 mmol) in DCM (0.5 mL) at RT. The reaction mixture wasstirred at RT overnight. The solvent was evaporated. The crude productwas purified by flash chromatography eluting from silica gel with agradient of 0-30% MeOH in DCM to afford 0.39 g of compound 25 as a whitesolid. MS: (+) m/z 371.1 (M+1).

Compound 26. N,N-methanediylidenedicyclohexanamine (DCC, 0.261 g, 1.267mmol) was added to a solution of compound 25 and1-hydroxypyrrolidine-2,5-dione (0.146 g, 1.267 mmol) in DCM (6 mL) atRT. After the reaction mixture was stirred at RT overnight, the solidwas filtered off. The filtrate was then concentrated. The crude productwas purified by flash chromatography eluting from silica gel with agradient of 0-100% EtOAc in hexanes to afford 0.43 g of compound 26 as acolorless oil.

Compound 27. DIEA was added to a solution of compound 21 (50 mg, 0.086mmol) and compound 26 (60.6 mg, 0.130 mmol) in DMF at RT, adjusting thepH to 8-9. After the reaction mixture was stirred at RT for 4 h, thereaction was quenched by addition of 10 mL of 1:1 mixture of 0.1% aq.TFA and acetonitrile. Preparative HPLC purification afforded 55 mg ofcompound 27 as a white solid, MS: (+) m/z 931.4 (M+1).

Compound 28. TFA (1 mL, 0.059 mmol) was added to a solution of compound27 (55 mg, 0.059 mmol) in DCM (2 mL) at RT. The reaction mixture wasstirred at RT for 10 min. The solvent was evaporated.

DIEA was added to a solution of compound 9 (32.7 mg, 0.059 mmol) andHATU (22.47 mg, 0.059 mmol) in DMF (1 mL). The pH of the reactionmixture was adjusted to 8-9. After the reaction mixture was stirred atRT for 10 min, DMF (2 mL) and DIEA were added. The pH of the reactionsolution was adjusted to 8-9. After the reaction mixture was stirred atRT for 10 min, LCMS showed the reaction was complete. The reaction wasquenched by addition of 20 mL 1:1 (v/v) mixture of water containing 0.1%TFA and acetonitrile. Preparative HPLC purification afforded 70 mg ofcompound 28 as a white solid. MS: (+) m/z 684.1 (M/2+1).

Compound (III-2). Piperidine was added to a solution of compound 28 (70mg, 0.051 mmol) in DMF (4 mL). After the reaction mixture was stirred atRT for 20 min, a 1:1 mixture of acetonitrile and 0.1% aq. TFA (40 mL)was added. Preparative HPLC purification afforded 46 mg of compound(III-2) as a white solid. MS: (+) m/z 1144.6 (M+1).

Compounds such as (III-2), having a hydroxylamine group, can be used toform conjugates with an antibody or other ligand having an aldehyde orketone functionality, for example by incorporation of the unnaturalamino acid 4-acetylphenylalanine.

Example 3 Compounds (I-2) and (I-3)

The synthesis of compounds (I-2) and (I-3) is shown schematically inFIG. 5.

Compound (I-3). DIEA was added to a solution of compound 9 (10 mg, 0.018mmol) and HATU (6.87 mg, 0.018 mmol) in DMF (0.3 mL), adjusting the pHto 8-9. After the reaction mixture was stirred at RT for 10 min,compound 29 (prepared according to Cheng et al. 2011, Example 17; 4.56mg, 0.018 mmol) in DMF (0.5 mL) and DIEA were added, adjusting pH to8-9. After the reaction mixture was stirred at RT for 20 min, thereaction was quenched by addition of 4 mL of 1:1 mixture of acetonitrileand 0.1% aq. TFA. Preparative HPLC purification afforded 12 mg ofcompound (I-3) (I-2) as a white solid. MS: (+) m/z 788.4 (M+1).

Compound (I-2). A mixture of compound (I-3) (12 mg, 0.015 mmol) andPd/C, 10 wt % (4 mg, 0.015 mmol) in methanol (0.5 mL) was stirred underan H₂ atmosphere overnight. The catalyst was filtered off, and thefiltrate concentrated. Preparative HPLC purification afforded 8.1 mg ofcompound (I-2) as a white solid. MS: (+) m/z 758.4 (M+1).

Compound (I-1) can be analogously prepared by replacing compound 29 withthe compound having mixed stereochemistry at the alpha-methyl position(Cheng et al. 2011).

Example 4 Compound (III-4)

FIGS. 6 a through 6 c in combination show schematically the synthesis ofcompound (III-4).

Compound 32. Compound 30 (Aldrich, 3.5 g, 13.9 mmol) was dissolved in 50mL DCM. To this solution was added Dess-Martin periodinane (11.8 g, 27.9mmol) at 5° C. After 10 min the mixture was warmed to RT. After anotherhour the reaction was quenched with saturated aq. NaHCO₃ and saturatedaq. NaS₂O₃. After extraction with ether, the ether extract was washedwith aq. NaHCO₃ and then brine and dried and evaporated down to a stickyoil. The oil was dissolved in 50 mL of DCM, to which was added thecommercially available compound 31 (5.05 g, 13.93 mmol). After 10 minthe reaction mixture was taken up in EtOAc, washed with aq. NaHCO₃ andthen brine and dried, filtered and the solvent evaporated. After columnchromatography (EtOAc: hexane, 0-20% gradient) compound 32 (2.3 g, 6.90mmol, 49.5% yield) was obtained as a white solid. It had an NMR spectrumconsistent with literature (Wipf et al. 2004a).

Compound 33. Hydrochloric acid (7.80 mL, 31.2 mmol, 4M in dioxane) wasadded at 5° C. to a DCM solution of compound 32 (5.2 g, 15.60 mmol).After the deprotective reaction was complete, the reaction mixture wasevaporated and compound 33 (4.21 g, 15.60 mmol, 100% yield,hydrochloride) was obtained as a white solid, which is used for nextstep without further purification.

Compound 35. To a solution of compound 34 (prepared per Sani et al.2007, 4.00 g, 11.6 mmol) in 20 mL DMF at 5° C. were added HATU (4.61 g,12.12 mmol) and DIPEA (6 ml, 34.4 mmol). After 10 min compound 33 (2.71g, 11.60 mmol) was added. After another half hour the mixture was takenup in EtOAc, which was washed with 10% aq. citric acid, saturatedaq.NaHCO₃ and brine. After drying and filtration, the organic phase wasevaporated down to give compound 35 (6.49 g, 11.60 mmol, 100% yield,[M+Na]⁺, calculated 582.3. found 582.3) as an oil, which was used fornext step without further purification.

Compound 36. NaBH₄ (4.66 g, 123 mmol) was added, in portions, to a 100mL methanol solution of compound 35 (6.49 g, 11.6 mmol) and NiSO₄(H₂O)₆(6.48 g, 24.66 mmol) at 5° C. (Caution: Hydrogen was generated.) After30 min, saturated aq. NaHCO₃ was added, followed by EtOAc. Afterfiltration through CELITE™, the organic phase was separated from theaqueous phase, washed with brine, dried, filtered and evaporated down togive compound 36 (5.6 g, 9.97 mmol, 81% yield, [M+1]⁺, calculated 562.3.found 562.4), which was used for next step without further purification.

Compound 37. Compound 36 (5.6 g, 9.97 mmol) was dissolved in 30 mLpyridine at 5° C. Acetic anhydride (4 g, 39.2 mmol) was added to thissolution. After 10 min the mixture was warmed to RT. After about an hourthe reaction mixture was concentrated down. The resulting residue wastaken up in EtOAC and the organic phase was washed with 10% aq. citricacid, saturated aq. NaHCO₃, and brine, sequentially. The organic phasewas dried, filtered and concentrated to give compound 37 (5.8 g, 9.61mmol, 100% yield, [M+1]⁺, calculated 604.3. found 604.4), which was usedfor next step without further purification.

Compounds 38a and 38b. Compound 37 (0.8 g, 1.3 mmol) was dissolved in 5mL methanol at −78° C. To this solution was added NaOMe (331 uL, 1.33mmol, 4M in MeOH). The mixture was allowed to warm up to RT over 1 hr.The mixture was taken up in EtOAc, washed with 10% aq. citric acid,saturated aq. NaHCO₃ solution, and brine. The separated organic phasewas dried, filtered and evaporated to give a mixture of ethyl and methylesters (compounds 38a and 38b, respectively). The mixture of esters wasnot separated during the next few steps, until both were hydrolyzed tothe carboxylic acid at a later step.

Compounds 40a and 40b. The mixture of compounds 38a and 38b from theabove reaction was dissolved in 20 mL DCM. To this solution was added4-nitrophenyl carbonochloridate 39 (524 mg, 2.6 mmol) and pyridine (210μl, 2.6 mmol) at 5° C. The temperature was allowed to rise to roomtemperature after 1 h and methylamine (1.950 mL, 3.9 mmol, 2M in THF)was added. After 10 min the solvent was evaporated and the residue waspassed through a chromatography column to give a mixture of compounds40a and 40b (ethyl and methyl esters, respectively; 420 mg, 40a/40bratio 3:1 from HPLC, about 53% yield for two steps, [M+1]⁺: calculated603.3. found 603.4 for 40a; calculated 589.3. found 589.4 for 40b).

Compounds 41a and 41b. The mixture of compounds 40a and 49b (420 mg,about 0.68 mmol) was dissolved in 3 mL DCM, to which was added HCl (4.8mmol, 1.2 mL, 4N in dioxane). After 1 h at 5° C. the solvent wasevaporated and the mixture of compounds 41a (ethyl ester) and 41b(methyl ester), ratio about 3:1, was used for next step without furtherpurification.

Compounds 43a and 43b. A mixture of compounds 41a and 41b (400 mg, ≈0.72mmol), compound 42 (commercially available from Anichem, 170 mg, 0.721mmol) and acetic acid (0.041 mL, 0.721 mmol) were mixed in DCM at 5° C.Sodium triacetoxyborohydride (306 mg, 1.44 mmol) was added. The mixturewas taken up in EtOAc after 30 min. After washed with 7% aq. K₂CO₃ andbrine, the organic phase was dried, filtered and evaporated down to givea residue. After column chromatography purification (MeOH: DCM, 0-7%gradient), compounds 43a (ethyl ester) and 43b (methyl ester) wereobtained (310 mg, approximately 0.42 mmol, approximately 58.3% yield,43a:43b ratio about 3:1, [M+1]⁺, calculated 738.4. found 738 for 43a;calculated 724.4. found 724 for 43b).

Compounds 45a and 45b. A mixture of compounds 43a and 43b (310 mg,approximately 0.42 mmol) was dissolved in 5 mL DCM at RT. To thissolution were added 2,6-di-tert-butylpyridine (161 mg, 0.840 mmol) and a2 mL DCM solution of compound 44 (prepared per Peltier et al. 2006, 73.8mg, 0.420 mmol). After half an hour Et₃N (58.6 μl, 0.420 mmol) wasadded. The mixture was then taken up in EtOAc, which was washed with 10%aq. citric acid, saturated aq. NaHCO₃ solution and brine. The organicphase was dried, filtered and evaporated down to a residue. After columnchromatography purification, compounds 45a and 45b were obtained (294mg, approximately 0.334 mmol, 80% yield, 45a:45b ratio 3:1, [M+1]⁺,calculated 877.5. found 877 for 45a; calculated 863.5 found 863 for 45b)as a sticky oil.

Compounds 46a and 46b. A mixture of compounds 45a and 45b (100 mg,approximately 0.114 mmol) was added to a suspension of Pd/C (65 mg, 10%)in 20 mL MeOH. HCl (28.5 μL, 0.114 mmol, 4M in dioxane) was added. Theflask was evacuated and refilled with H₂, this process being repeatedthree times. After 2 h the suspension was filtered and the solvent wasevaporated to give a residue. A suspension of compound 5a (19.59 mg,0.137 mmol) in 500 uL DMF, HATU (43.4 mg, 0.114 mmol) and DIPEA (49.8μL, 0.285 mmol) were added at 5° C. After the suspension becamehomogeneous the above residue was added as a DMF (1 mL) solution. MoreDIPEA was added to adjust the pH to about 12. After 10 min the mixturewas taken up in EtOAc, which was washed with 10% aq. citric acid,saturated aq. NaHCO₃ and brine. The separated organic phase was dried,filtered and evaporated. The resulting residue was passed through achromatographic column to give a mixture of compounds 46a and 46b (ethyland methyl esters, respectively, 80 mg, about 0.082 mmol, 71.9% yield,46a:46b ratio 3:1, [M+1]⁺, calculated 976.5. found 976.5 for 46a;calculated 962.5. found 962.5 for 46b).

Compounds 47a and 47b. HCl (256 μl, 1.024 mmol, 4M in dioxane) was addedto a 2 mL MeOH solution of compounds 46a and 46b (200 mg, 0.205 mmol) at5° C. After 1 h the solution was evaporated down and dried on highvacuum overnight to give a sticky oil. This sticky oil, Fmoc-protectedcitrulline 18a (81 mg, 0.205 mmol), and DIPEA (179 μl, 1.024 mmol) weredissolved in 2 mL DMF at RT. Propylphosphonic acid anhydride (T3P, 178μL, 0.410 mmol, 2.3 M in EtOAc) was added. After 1 h the reactionmixture was taken up in EtOAc, which was washed with saturated aq.NaHCO₃ solution and brine. After separation, drying and evaporation, theresulting residue was passed through a chromatography column (MeOH: DCM,0-10% gradient) to give a mixture of compounds 47a and 47b (ethyl andmethyl esters, respectively, 150 mg, approximately 0.119 mmol, about58.3% yield, 47a:47b ratio 3:1, [M+1]⁺, calculated 1255.6. found 1255.6for 47a; calculated 1241.6. found 1241.6 for 47b).

Compound 49. A mixture of compounds 47a and 47b (200 mg, about 0.165mmol) was dissolved in 5 mL of DMF (with 5% piperidine) at roomtemperature. The solution was evaporated to dryness after 30 min. Theresulting residue was mixed with Boc-protected valineN-hydroxysuccinimide ester 48 (61.9 mg, 0.198 mmol), 5 mL DMF and DIPEA(87 μL, 0.496 mmol). After allowing reaction to proceed overnight, thereaction mixture was evaporated to dryness. The resulting mixture wasdissolved in a 5 mL mixture of MeOH, THF and water (1:1:1). NaOH wasadded and the pH of the final solution was 14. After allowing reactionto proceed overnight at RT, the mixture was acidified with HCl to a pHof 3 and evaporated under high vacuum. The obtained solid was treatedwith TFA and the mixture was evaporated after 10 min, affording compound49 (80 mg, 0.072 mmol, 43.8% yield, [M+1]⁺, calculated 1104.6. found1104.6) after preparative HPLC purification.

Compound (III-4). Compound 49 (80 mg, 0.072 mmol), commerciallyavailable compound 21a (Aldrich, 26.6 mg, 0.087 mmol) and DIPEA (38.0μl, 0.217 mmol) were dissolved in 2 mL of DMF. After reaction wasallowed to proceed overnight, the mixture was evaporated down and theresidue was purified by preparative HPLC to give a major isomer (15 mg,16% yield, ½[M+2]²⁺, calculated 649.3. found 649.5) and a minor isomer(3.7 mg, 4% yield, ½[M+2]²⁺, calculated 649.3. found 649.5)). The majorisomer was tentatively assigned as (III-4), having the natural tubulysinstereochemistry at the alpha-methyl of the Tup subunit and the minorisomer was assigned as compound (III-5), having the invertedstereochemistry there.

Example 5 Compounds (I-5), (I-6), and (I-7)

A small portion of the sticky oil described above from the treatment ofcompounds 46a and 46b with HCl was not coupled to compound 18a but wasinstead dissolved in a mixture of THF, MeOH and water (1:1:1). The pH ofthe reaction mixture was adjusted to 14. After allowing the reaction toproceed overnight, half of the reaction mixture was evaporated andpurified by preparative chromatography to give compounds (I-5) (1 m;M+1, 876.6), (I-6) (1 mg; M+1, 862.5) and (I-7) (1 mg; M+1, 848.5).

Example 6 Compound (I-1)

A scheme for the synthesis of compound (I-1) is shown in FIG. 7.

Compound 51. HCl (6 N, 0.2 mL) was added to a solution of compound 50(Cheng et al. 2011; 50 mg, 0.198 mmol) and 2,2-dimethoxypropane (0.244mL, 1.982 mmol) in MeOH (1 mL). After the reaction mixture was stirredat RT overnight, the solvent was evaporated to afford 52.8 mg ofcompound 51. MS: (+) m/z 267.2 (M+1).

Compound 52. A mixture of compound 51 (52.8 mg, 0.198 mmol) andpalladium on carbon (10 wt %, 8 mg) in MeOH (1 mL) was stirred under anH₂ atmosphere overnight. The catalyst was then filtered off and thesolvent evaporated to afford 46.9 mg of compound 52. MS: (+) m/z 237.3(M+1).

Compound (I-1). A mixture of pentafluorophenol (2.493 mg, 0.014 mmol),1,3-dicyclohexylcarbodiimide (2.049 mg, 9.93 μmol), and compound 9 (5mg, 9.03 μmol) in DCM (0.5 mL) was stirred at RT overnight. The solventwas then evaporated.

To a solution of the resulting residue (6.50 mg, 9.03 μmol) and compound52 (4.27 mg, 18.06 μmol) in DMF (0.2 mL) was added DIEA (1 drop). Afterthe reaction mixture was stirred at RT for 10 min, the reaction wasquenched by addition of a 1:1 mixture of acetonitrile and watercontaining 0.1% TFA (4 mL). Preparative HPLC purification afforded 2.5mg of compound (I-1) as a white solid. MS: (+) m/z 772.5 (M+1).

Example 6 Compound (I-4)

FIG. 8 shows a scheme for the synthesis of compound (I-4).

DIEA was added to a solution of compound 9 (10.69 mg, 0.019 mmol) andHATU (7.34 mg, 0.019 mmol) in DMF (0.3 mL), adjusting pH to 8-9. Afterthe reaction mixture was stirred at rt for 10 min, compound 53 (4 mg,0.019 mmol; prepared according to Sani et al. 2007) in DMF (0.5 mL) andDIEA were added, adjusting the pH to 8-9. After the reaction mixture wasstirred at RT for 20 min, the reaction was quenched by addition of a 1:1mixture of acetonitrile and water containing 0.1% TFA (4 mL).Preparative HPLC purification afforded 12.5 mg of compound (I-4) as awhite solid. MS: (+) m/z 743.4 (M+1).

Example 7 Thiocarbamates

Compounds according to formula (I) in which W is S (that is,thiocarbamates) can be made by treating a suitable precursor such ascompound 6 (FIG. 1) with sodium hydride and then a thioisocyanate, asillustrated below:

Example 8 Compound (III-6)

A scheme for the synthesis of compound 56 is shown in FIGS. 9 a and 9 b.

Compound 56. 1,3-Dicyclohexylcarbodiimide (DCC, 0.160 g, 0.778 mmol) wasadded to a solution of tert-butyl1-amino-3,6,9,12-tetraoxapentadecan-15-oate 54 (Quanta Biosciences, 0.25g, 0.778 mmol) and2-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-oxy)acetic acid 55(Chem-Impex, 0.244 g, 0.778 mmol) in DCM (5 mL) at RT. After thereaction mixture was stirred at RT overnight, the precipitate wasfiltered off. The filtrate was then concentrated. The crude product waspurified by flash chromatography eluting from silica gel with a gradientof 0-10% methanol in DCM to afford 0.30 g of compound 56 as a colorlessoil. MS: (+) m/z 617.4 (M+1).

Compound 57. A solution of compound 56 (0.303 g, 0.491 mmol) in TFA (2mL, 0.662 mmol) was stirred at RT for 2 h. After the solution wasconcentrated, the residue was washed with hexanes to afford 0.28 g ofcompound 57. MS: (+) m/z 561.3 (M+1).

Compound 58. DCC (0.199 g, 0.963 mmol) was added to a solution ofcompound 57 (0.27 g, 0.482 mmol) and 1-hydroxypyrrolidine-2,5-dione(also known as N-hydroxysuccinimide or NHS, 0.111 g, 0.963 mmol) in DCM(5 mL) at RT. After the reaction mixture was stirred at RT overnight,the solid was filtered off. The filtrate was then concentrated. Thecrude product was purified by flash chromatography eluting from silicagel with a gradient of 0-100% ethyl acetate in hexanes to afford 0.12 gof compound 58 as a colorless oil. MS: (+) m/z 658.3 (M+1).

Compound 59. DIEA (2 drops) was added to a solution of compound 58 (0.12g, 0.182 mmol) and compound 21 (0.106 g, 0.182 mmol) in DMF (2 mL) atRT. After the reaction mixture was stirred at RT for 1 h, the reactionwas quenched by addition of a mixture of acetonitrile and watercontaining 0.1% TFA. The crude product was purified by prep HPLC toafford 0.12 g of compound 59 as a white solid. MS: (+) m/z 1121.6 (M+1).

Compound 60. TFA (0.5 mL) was added to a solution of compound 59 (20 mg,0.018 mmol) in DCM (1 mL) at RT. After the reaction mixture was stirredat RT for 20 min, the solution was concentrated to afford 18.2 mg ofcompound 60. MS: (+) m/z 1021.6 (M+1).

Compound 61. DIEA was added to a solution of compound 9 (9.87 mg, 0.018mmol) and HATU (6.78 mg, 0.018 mmol) in DMF (0.4 mL). The pH of thereaction mixture was adjusted to 8-9. After the reaction mixture wasstirred at RT for 10 min, compound 60 (18.2 mg, 0.018 mmol) in DMF (1mL) and DIEA were added. The pH of the reaction mixture was adjusted to8-9. After the reaction mixture was stirred at RT for 10 min, thereaction was quenched by addition of 10 mL 1:1 (v/v) mixture of watercontaining 0.1% TFA and acetonitrile. The crude product was purified byprep HPLC to afford 25 mg of compound 61 as a white solid. MS: (+) m/z779.0 (M/2+1).

Compound (III-6). One drop of piperidine was added to a solution ofcompound 61 (25 mg, 0.016 mmol) in DMF (1 mL) at RT. After the reactionmixture was stirred at RT for 1 h, the reaction was quenched by additionof a mixture of acetonitrile and water containing 0.1% TFA. The crudeproduct was purified by prep HPLC to afford 20 mg of compound (III-6) asa white solid. MS: (+) m/z 668.0 (M/2+1). Compound (III-6) has ahydroxylamine group, which can be use for conjugation via oximeformation, for instance with the ketone group of a p-acetylphenyalanineresidue that has been introduced into a protein, as discussed above.

A conjugate of compound (III-6) and an anti-mesothelin antibody modifiedto contain a p-acetylphenylalanine residue exhibited an EC₅₀ of 0.14against N87 gastric cancer cells, using a ³H thymidine incorporationassay.

In one embodiment, this invention provides a conjugate of compound(III-6) and an anti-mesothelin antibody modified to contain a ketonegroup. Preferably, the modification is by the incorporation of ap-acetylphenylalanine residue into the antibody polypeptide chain. Alsopreferably, the antibody so modified is 6A4.

Example 9 Intermediate Compound 69

FIG. 10 shows a scheme for the synthesis of compound 69, which can beused as an intermediate for the synthesis of compounds of thisinvention.

Compound 63. Compound 62 (Chem-Impex, 5 g, 23.8 mmol) was added into asolution of SOCl₂ (3.47 mL, 47.6 mmol) in 20 mL MeOH at 5° C. Afterallowing reaction to proceed overnight, the reaction mixture was heatedfor half an hour at 45° C. The volatile materials were evaporated andthe residue was dissolved in 20 mL DCM. Boc₂O (7.8 g, 35.7 mmol) wasadded. Et₃N was used to adjust the pH of the solution to 9 (tested withmoistened pH paper). After a few hours the reaction mixture was taken upin EtOAc. The EtOAc solution was washed with 10% citric acid solution,sat. aq. NaHCO₃ solution and brine. The organic phase was dried,separated and evaporated down. The final residue from the evaporation ofthe dried organic phase was passed through a column to give compound 63(5 g, 65% yield, [M+1-Boc]⁺, calculated 225.1. found 225.2).

Compound 65. To a 20 mL DCM solution of compound 63 (2 g, 6.2 mmol) wasadded DIBAL-H (12.4 mL, 12.4 mmol, 1M in DCM) at −78° C. After half anhour, MeOH was used to quench the reaction. HCl was used to adjust thepH of the solution to around 2. The mixture was taken up in EtOAc, whichwas washed with 10% citric acid, brine, dried, separated and evaporated.The residue was dissolved in 30 mL of DCM. Compound 64 (2.4 g, 6.2 mmol,U.S. Pat. No. 4,894,386) was added at 0° C. The mixture was evaporatedafter 1 h at RT, the residue was passed through a column to givecompound 65 (2 g, 79% yield, [M+1-Boc]⁺, calculated 307.2. found 307.1).

Compound 66. Compound 65 (600 mg, 1.48 mmol) was dissolved in 75 mLEtOAc at RT. The solution was transferred a flask filled with N₂ andPd/C (600 mg, 10%). The flask was evacuated and refilled with H₂; thecycle was repeated three times. After 1 h the solution was filtered andevaporated to give compound 66 (560 mg, 100% yield, [M+1]⁺, calculated379.3. found 379.3) as a mixture of epimers. This mixture was notseparated until later.

Compound 67. Compound 66 (1.30 g, 3.43 mmol), Fmoc-protected citrulline18a (1.6 g, 4.12 mmol) and2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 1.06 g, 4.3 mmol)were dissolved in a mixture of DCM and MeOH (22 mL, 10:1). Afterallowing reaction to proceed overnight, the mixture was evaporated andthe residue was passed through a column (MeOH:DCM, 0-5% gradient) togive compound 67 ([M+1]⁺, 7calculated 758.4. found 758.4) as a mixtureof epimers (=4:1 from HPLC analysis). It was used for next step withoutfurther purification.

Compound 68a. The mixture of compound 67 from the above reaction wasdissolved in 10 mL of DMF (with 5% piperidine) at RT. After 30 min, themixture was evaporated and dried with a high vacuum pump overnight. Theresidue was mixed with compound 48 (1.5 g, 3.45 mmol) in 5 mL DMF. Et₃Nwas used to adjust the pH of the solution to 12. After 1 h the reactionmixture was taken up in EtOAc, which was washed with 10% citric acid,sat. aq. NaHCO₃ solution and brine. The organic phase was dried,filtered and evaporated to dryness. The residue was deprotected with 5%piperidine in DMF in the same way as the deprotection of compound 67.The amine obtained in this step and compound 21a (1 g, 3.27 mmol) weremixed in 10 mL of DMF. Et₃N was used to adjust the pH of the solution to12 at RT. After allowing the reaction to proceed overnight, the mixturewas taken up in EtOAc, which was washed with 10% citric acid, sat. aq.NaHCO₃ and brine. The organic phase was dried, filtered and evaporatedto give a residue. The residue was passed through a regular silicacolumn to give a mixture of compounds 68a and 68b (1 g, 35% yield fromcompound 66, [M+1]⁺, calculated 828.5. found 828.5). After separation ona preparative HPLC column, 600 mg of the major epimer was obtained(structure tentatively assigned as compound 68a, with compound 68bassigned as the minor one).

Compound 69. Compound 68a (600 mg, 0.72 mmol) was dissolved in a 5 mLmixture of DCM and TFA (1:1). After 2 h, the reaction mixture wasevaporated to give compound 69 (quantitative yield, [M+1], calculated672.4. found 672.4).

Example 10 Intermediate Compound 75

FIG. 11 shows a scheme for the synthesis of intermediate compound 75,which can be used for the synthesis of compounds of this invention.

Compound 71. Compound 70 (Cheng et al. 2011, 25 mg, 0.044 mmol),N,N′-diisopropylcarbodiimide (DIC, 0.014 mL, 0.088 mmol),4-(dimethylamino)pyrdine (DMAP, 10.78 mg, 0.088 mmol) and MeOH (0.036mL, 0.882 mmol) were mixed at RT. After an hour, the reaction mixturewas evaporated and passed through a column to give compound 71 (10 mg,39% yield, M+1, 581.4).

Compound 72. Compound 71 (122 mg, 0.210 mmol, from another syntheticbatch) was dissolved in MeOH (2 mL) at 5° C. NaOMe (0.441 mL, 0.221mmol) was added. After 0.5 h the mixture was neutralized with HCl (4M indioxane) and evaporated to give compound 72 (m+1, 539.4), which was usedfor next step without further purification.

Compound 73. Compound 72 (60 mg, 0.111 mmol) was dissolved in DCM (1.5mL) at 5° C. Pyridine (0.045 mL, 0.557 mmol).4-Nitrophenylcarbonochloridate 72a (67.3 mg, 0.334 mmol, Aldrich) in 0.5mL DCM was added slowly. After allowing reaction to proceed overnight,the mixture was evaporated and purified by column chromatography to givecompound 73 (42 mg, 0.060 mmol, 53.6% yield) (m+1, 704.4).

Compound 74. Compound 73 (42 mg, 0.060 mmol) was dissolved in DCM (1 mL)at 5° C. Methylamine (1.853 mg, 0.060 mmol) was added. After 0.5 h themixture was evaporated and the residue was passed through achromatographic column (MeOH:DCM, 0-15% gradient, product eluting out at7-10%) to give compound 74 (35 mg, 0.059 mmol, 98% yield) (m+1, 596.4).

Compound 75. Compound 74 (93 mg, 0.156 mmol) was dissolved in THF (1 mL)at 5° C. LiOH (7.47 mg, 0.94 mmol) in 0.34 mL water was added. After thereaction finished, the reaction mixture was neutralized with HCl (4M indioxane) and dried on high vacuum to give compound 75 (m+1, 582.3),which was used for next step without further purification.

Example 11 Compounds (III-7) and (III-8)

FIG. 12 shows schemes for the synthesis of compounds (III-7) and(III-8).

Compound (III-7).

Compound 75 (11 mg, 0.019 mmol) was activated in DMF (0.5 mL) with HATU(6.83 mg, 0.018 mmol) and DIEA (14.86 μl, 0.085 mmol). Compound 69(15.24 mg, 0.023 mmol) was then added. After 10 min the reaction mixturewas taken up in DMSO and purified by preparative chromatography to givecompound (III-7) (12 mg, 9.71 μmol, 51.4% yield) (m+1, 1235.7).

Compound (III-8). Compound 75 (10 mg, 0.017 mmol) was activated in DMF(0.5 mL) with HATU (6.21 mg, 0.016 mmol) and DIEA (0.014 mL, 0.077mmol). Compound 23 (13.86 mg, 0.021 mmol) was then added. After 10 minthe reaction mixture was taken up in DMSO and purified by preparativechromatography to give compound (III-8) (13 mg, 10.52 μmol, 61.2% yield)(m+1, 1235.7).

Example 12 Compounds (I-8) and (I-9)

In principle, compounds (I-8) and (I-9) can be obtained by treatment ofcompounds (III-7) and (III-8), respectively, with the protease cathepsinB, for which the dipeptide Val-Cit is a substrate motif. Compound(III-7), with its ortho-amino group, may be subject to more sterichindrance against cleavage.

Example 13 Biological Activity of Compounds

FIGS. 13 a and 13 b show the biological activity of compounds (I-4) and(I-2) of this invention against H226 lung cancer and 786-O renal cancercells, respectively, using an ATP luminescence assay. As controls,doxorubicin and the tubulysin analog Compound A, which contains anacetate group instead of a carbamate group at the Tuv subunit, wereused. Compound A can be prepared according to the teachings of Cheng etal. 2011.

Against H226 cells, the EC₅₀ values were: doxorubicin, 115.4 nM;Compound A, 2.4 nM; and compound (I-4), 12.1 nM. Against 786-O cells,the EC₅₀ values were: doxorubicin, 68.9 nM; Compound A, 1.2 nM, andcompound (I-4), 7.1 nM.

FIGS. 13 c and 13 d present the similar type of data for compounds(I-5), (I-6), and (I-7). The comparison compounds were doxorubicin andtubulysin D. The EC₅₀ values against H226 cells were: doxorubicin, 115.4nM; tubulysin D, 0.05 nM; compound (I-5), 19.5 nM; compound (I-6), 9.9nM; and compound (I-7), 15.4 nM. Against 786-O cells, the EC₅₀ valueswere: doxorubicin, 68.9 nM; tubulysin D, 0.02 nM; compound (I-5), 22.9nM; compound (I-6), 22.8 nM; and compound (I-7), 12.5 nM.

Tumor cell lines were obtained from the American Type Culture Collection(ATCC), P.O. Box 1549, Manassas, Va. 20108, USA, and cultured accordingto ATCC instructions. Cells were seeded at 1.0×10³ cells/well in 96-wellplates for 3 h for the ATP assays. 1:3 serial dilutions of the compoundswere added to the wells. Plates were allowed to incubate for 24 to 72 h.ATP levels in the ATP plates were measured using the CELLTITER-GLO®Luminescent Cell Viability kit following the manufacturer's manual andread on a GLOMAX® 20/20 luminometer (both from Promega, Madison, Wis.,USA). The EC₅₀ values—the concentration at which an agent inhibits orreduces cell proliferation by 50%—were determined using PRISM™ software,version 4.0 (GraphPad Software, La Jolla, Calif., USA).

Example 14 In Vitro Activity of a Conjugate

FIG. 14 shows the in vitro activity of conjugate (II-1), against N87gastric cancer cells (American Type Culture Collection (ATCC), P.O. Box1549, Manassas, Va. 20108, USA).

Cells were seeded at 1.0×10⁴ cells/well in 96-well plates for 3 h for ³Hthymidine assays, respectively. Serial dilutions (1:3) of conjugate(II-1) were added to the wells. Plates were allowed to incubate for 120h. The plates were pulsed with 1.0 μCi of ³H-thymidine per well for thelast 24 h of the total incubation period, harvested, and read on a TopCount Scintillation Counter (Packard Instruments, Meriden, Conn.). TheEC₅₀ value—the concentration at which an agent inhibits or reduces cellproliferation by 50% of the maximum inhibition—was determined usingPRISM™ software, version 4.0 (GraphPad Software, La Jolla, Calif., USA)to be 0.2 nM

A comparison of the EC₅₀ values from FIGS. 13 a-13 d and FIG. 14illustrates two points. First is the potency enhancement associated withthe targeted delivery of a cytotoxin via a conjugate and then the activeinternalization mechanism triggered by binding of the antibody componentof the conjugate to its antigen (Schrama et al. 2006). Second, theunconjugated toxins are relatively polar compounds and, in the absenceof an active internalization mechanism, have difficulty diffusing acrossa cell membrane, thus resulting in higher (lower potency) measured EC₅₀values.

Example 15 In Vivo Activity of a Conjugate

In this example, the in vivo activity of conjugate (II-1) was comparedagainst that of Conjugate B (Cheng et al. 2011), which is structurallyidentical to conjugate (II-1), except that it has an acetate in the Tuvsubunit instead of a carbamate:

Five million OVCAR3 ovarian cancer cells, resuspended in 0.1 mLphosphate buffered saline (“PBS”) plus 0.1 mL matrigel, were implantedsubcutaneously at the flank region of SCID mice. Tumor measurementsstarted 28 days later, and mice were randomized into groups of 7 miceeach with average tumor sizes of 60 mm³ estimated by LWH/2 of tumors. At29 days post tumor implantation, mice were dosed intraperitoneallysingly with testing compounds. FIG. 15 shows that, against OVCAR3xenografts, conjugate (II-1) suppressed tumor growth more effectivelythan conjugate B. The differential is especially notable after 20 days.

FIGS. 16 a, 16 b, 17 a, 17 b, 18 a, 18 b, 19 a, 19 b, and 20 presentadditional in vivo efficacy data for conjugates of this invention,generated per the protocol described above, mutatis mutandis.

FIG. 16 a shows the data for OVCAR3 (ovarian cancer) tumor volume versustime for a series of conjugates of compound (III-1) with anti-CD70antibody 1F4 or anti-mesothelin antibody 6A4. The legend provides, inorder, the DAR (e.g., “2.7”) and dosage in μmol/kg (e.g., “0.1”). Ineach instance the mode of administration was intraperitoneal, in asingle dose (SD), except for last data set (♦), for which the conjugatewas administered Q7Dx3. FIG. 16 b shows the body weight change for thesame experiment.

The preparation and characterization of human monoclonal antibody 6A4 isdescribed in Terrett et al. 2012, the disclosure of which isincorporated herein by reference. The V_(H) CDR1, CDR2, and CDR3 andV_(K) CDR1, CDR2, and CDR3 sequences for antibody 6A4 are given in SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ IDNO:6, respectively. The variable region V_(H) and V_(K) sequences ofantibody 6A4 are provided in SEQ ID NO:7 and SEQ ID NO:8, respectively.

The preparation and characterization of human monoclonal antibody 1F4 isdescribed in Coccia et al. 2010, the disclosure of which is incorporatedherein by reference. The V_(H) CDR1, CDR2, and CDR3 and V_(K) CDR1,CDR2, and CDR3 sequences for antibody 1F4 are given in SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14,respectively. The variable region V_(H) and V_(K) sequences of antibody1F4 are provided in SEQ ID NO:15 and SEQ ID NO:16, respectively.

FIGS. 17 a and 17 b present the results for a similar experiment, butwith conjugates of compound (III-8) and anti-mesothelin antibody 6A4. Ineach instance, a single dose was administered intraperitoneally.

The efficacy of a conjugate of compound (III-1) and anti-mesothelinantibody 6A4 against H226 (lung cancer) tumors is presented in FIGS. 18a and 18 b, with DAR and dosage information found again in the legends.In each instance the conjugate was administered intraperitoneally in asingle dose.

FIGS. 19 a and 19 b present H226 data for a conjugate of compound(III-8) and anti-mesothelin antibody 6A4. In the first data set (●) theadministration was a single dose, while for the last two data sets (▾and ♦) the administration regimen was Q7Dx3.

FIG. 20 shows the efficacy data for conjugates of compound (III-1) withanti-mesothelin antibody 6A4 or anti-CD70 antibody 1F4 against N87gastric cancer tumors.

Antibodies, especially when used in conjugates, may have either anatural constant region or an engineered (modified) one, designed toreduce or eliminate effector functions such as ADCC. Examples of suchmodified antibody constant regions are provided by the polypeptides ofSEQ ID NO:25 and SEQ ID NO:26. SEQ ID NO:25 is a constant region of theIgG4 isotype modified with certain amino acid substitutions. SEQ IDNO:26 is a hybrid IgG1/IgG4 constant region. In both SEQ ID NO:25 andSEQ ID NO:26 the presence of the C-terminal lysine is optional.

Example 16 Stability Studies

In this example, the mouse serum stability of Conjugate (II-1) andConjugate B, with their respective carbamate and acetate groups, werecompared.

The conjugate was injected into mice at a dose of 0.1 μmol/kg. In thecase of conjugate (II-1) the administration concentration was 3.4 mg/mLand the conjugate had a substitution ratio (SR) of 4.2. In the case ofconjugate B the administration concentration was 1.2 mg/mL and the SRwas 2.3. Approximately 100 uL of serum from each of 3 animals at eachtime point was taken for analysis.

Serum samples from the different animals were pooled, giving 200-300 uLat each time point. The pooled volume was centrifuged to remove solidsand the supernatant was used for analysis. The conjugate was isolatedfrom serum by immuno-affinity capture using an anti-idiotypic monoclonalantibody coupled to SEPHAROSE™ beads. After capture, the conjugate waseluted by exposure to low pH followed by neutralization with Tris base.Cytotoxin present on the conjugate was released by addition of activatedcathepsin B to cleave the Cit-Val peptide linker. Cathepsin B digestionwas done at 37° C. for 3 h, followed by addition of 1 volume of coldmethanol. The solvent-extracted cytotoxin was analyzed by LC-MS using anESI-TOF MS in-line to a UPLC with reverse phase chromatography (AcquityHSS T3 2.1×50 mm).

For conjugate (II-1), the presence of the hydroxyl compound fromhydrolysis of the carbamate group was not detected at any time, throughtime points up to 240 h. Only the carbamate compound was detected.

Conversely, for conjugate B, the hydrolysis product was detectable after6 hours and about 50 per cent hydrolysis had occurred by 72 h. Table 2below shows the relative amounts of the acetate and hydroxyl compounds,based on mass spectrum intensities for the respective doubly chargedions (M[H²⁺] 372.2 Da and 351.2 Da):

TABLE 2 Time Relative Peak Intensity Hydroxyl Ion (h) Acetate IonHydroxyl Ion (% of total) 0.25 360,587 — 0 2 649,982 — 0 6 165,68011,779 7 24 77,110 14,619 16 48 26,760 16,312 38 72 18,243 17,124 48 1687,978 18,330 70 240 4,268 15,654 79

These results show that while replacement of the acetate group in theTuv subunit leads to a much more stable compound, which however stillretains substantial biological activity.

Example 17 Compounds 72a, 72b, and 72c

This example describes the preparation and properties of compounds ofthis invention having structural variations at the carbamate group.Reference is made to FIG. 21 for the synthetic scheme.

Compound 70a. Ammonia in MeOH (2M, 0.089 mL, 0.178 mmol) was added tocompound 7 (0.1 g, 0.148 mmol) in MeOH (2 mL). After the reactionmixture was stirred at RT for 20 min, LCMS showed the reaction went tocompletion. The solvent was evaporated. The crude product was purifiedby flash chromatography eluting from silica gel with a gradient of 0-15%MeOH in DCM to afford 55 mg of compound 70a as a white solid. MS: (+)m/z 554.3 (M+1).

Compound 71a LiOH (4.41 mg, 0.184 mmol) in water (0.5 mL) was added to asolution of compound 70a (51 mg, 0.092 mmol) in THF (1 mL) at RT. Afterthe reaction mixture was stirred at RT for 2 h, LCMS showed the reactionhad gone to completion. The solvent was evaporated. The crude productwas purified by flash chromatography eluting from silica gel with agradient of 0-30% MeOH in DCM to afford 47 mg of compound 71a as a whitesolid. MS: (+) m/z 540.3 (M+1).

Compound 72a. DIEA (6.45 μL, 0.037 mmol) was added to a solution ofcompound 71a (20 mg, 0.037 mmol) and HATU (14.09 mg, 0.037 mmol) in DMF(0.5 mL). The pH of the reaction mixture was adjusted to 8-9. After thereaction mixture was stirred at RT for 20 min, compound 23 (24.9 mg,0.037 mmol) in DMF (1 mL) and DIEA (6.45 μL, 0.037 mmol) were added. ThepH of the reaction solution was adjusted to 8-9. After the reactionmixture was stirred at RT for another 20 min, LCMS showed the reactionwent to completion. The reaction was quenched by the addition of 10 mL1:1 (v/v) mixture of water containing 0.1% TFA and acetonitrilecontaining 0.1% TFA. The crude product was purified by preparative HPLCto afford 35.2 mg of compound 72a as a white solid. MS: (+) m/z 1193.6(M+1). Compound 72a is also referred to herein above as compound(III-9).

A conjugate of compound 72a and anti-CD70 antibody 1F4 had an EC₅₀ of0.19 nM against 786-O renal cancer cells. A conjugate of compound 72aand anti-mesothelin antibody 6A4 had an EC₅₀ of 0.16 nM against N87gastric cancer cells. In both instances, an ³H-thymidine incorporationassay was used.

Compound 70b. A solution of compound 7 (0.1 g, 0.148 mmol) and aniline(0.041 mL, 0.444 mmol) in DMF (1.8 mL) was heated at 50° C. overnight.The solvent was evaporated. The crude product was purified by flashchromatography eluting from silica gel with a gradient of 0-15% MeOH inDCM to afford 58 mg of compound 5 as a white solid. MS: (+) m/z 630.4(M+1).

Compound 71b. LiOH (4.03 mg, 0.168 mmol) in water (0.5 mL) was added toa solution of compound 70b (53 mg, 0.084 mmol) in THF (1 mL) at RT.After the reaction mixture was stirred at RT for 2 h, LCMS showed thereaction went to completion. The solvent was evaporated. The crudeproduct was purified by flash chromatography eluting from silica gelwith a gradient of 0-30% MeOH in DCM to afford 43 mg of compound 71b asa white solid. MS: (+) m/z 616.3 (M+1).

Compound 72b. DIEA (5.66 μL, 0.032 mmol) was added to a solution ofcompound 71b (20 mg, 0.032 mmol) and HATU (12.35 mg, 0.032 mmol) in DMF(0.5 mL). The pH of the reaction mixture was adjusted to 8-9. After thereaction mixture was stirred at RT for 20 min, compound 23 (21.82 mg,0.032 mmol) in DMF (1 mL) and DIEA (5.66 μL, 0.032 mmol) were added. ThepH of the reaction solution was adjusted to 8-9. After the reactionmixture was stirred at RT for another 20 min, LCMS showed the reactionwent to completion. The reaction was quenched by the addition of 10 mL1:1 (v/v) mixture of water containing 0.1% TFA and acetonitrilecontaining 0.1% TFA. The crude product was purified by preparative HPLCto afford 35 mg of compound 72b as a white solid. MS: (+) m/z 1269.7(M+1). Compound 72b is also referred to hereinabove as compound(III-10).

A conjugate of compound 72b and anti-CD70 antibody 1F4 had an EC₅₀ of0.58 nM against 786-O renal cancer cells. A conjugate of compound 72band anti-mesothelin antibody 6A4 had an EC₅₀ of 0.47 against N87 gastriccancer cells. In both instances a ³H thymidine incorporation assay wasused.

Compound 70c. Dimethylamine in DMF (1 mL, 0.148 mmol) was added dropwiseto a solution of compound 7 (0.1 g, 0.148 mmol) in DMF (1 mL) at RT,until the reaction went to completion. The solvent was evaporated. Thecrude product was purified by flash chromatography eluting from silicagel with a gradient of 0-15% MeOH in DCM to afford 56 mg of compound 70cas a white solid. MS: (+) m/z 582.3 (M+1).

Compound 71c. LiOH (8.23 mg, 0.344 mmol) in water (0.5 mL) was added toa solution of compound 70c (0.1 g, 0.172 mmol) in THF (1 mL) at RT.After the reaction mixture was stirred at RT for 2 h, LCMS showed thereaction went to completion. The solvent was evaporated. The crudeproduct was purified by flash chromatography eluting from silica gelwith a gradient of 0-30% MeOH in DCM to afford 50.8 mg of compound 71cas a white solid. MS: (+) m/z 568.3 (M+1).

Compound 72c. DIEA (6.14 μL, 0.035 mmol) was added to a solution ofcompound 71c (20 mg, 0.035 mmol) and HATU (13.39 mg, 0.035 mmol) in DMF(0.5 mL). The pH of the reaction mixture was adjusted to 8-9. After thereaction mixture was stirred at RT for 20 min, compound 23 (23.67 mg,0.035 mmol) in DMF (1 mL) and DIEA (6.14 μL, 0.035 mmol) were added. ThepH of the reaction solution was adjusted to 8-9. After the reactionmixture was stirred at RT for another 20 min, LCMS showed the reactionwent to completion. The reaction was quenched by the addition of 10 mL1:1 (v/v) mixture of water containing 0.1% TFA and acetonitrilecontaining 0.1% TFA. The crude product was purified by preparative HPLCto afford 23.8 mg of compound 72c as a white solid. MS: (+) m/z 1221.6(M+1). Compound 72c is also referred to hereinabove as compound(III-11).

A conjugate of compound 72c and anti-CD70 antibody 1F4 had an EC₅₀ of0.32 nM against 786-O renal cancer cells. A conjugate of compound 72cand anti-mesothelin antibody 6A4 had an EC₅₀ of 0.34 nM against N87gastric cancer cells. In both instances a ³H thymidine incorporationassay was used.

Example 18 Compounds 75a, 75b, and 75c

This example describes the preparation of the tubulysin analogs of theprevious example, but without the linker moieties attached. Reference ismade to FIG. 22 for the synthetic scheme.

Compound 73. LiOH (0.071 g, 2.97 mmol) in water (5 mL) was added to asolution of compound 18 (0.25 g, 0.743 mmol) in THF (5 mL) at RT. Afterthe reaction mixture was stirred at RT for 2 h, the solvent wasevaporated. The crude product was purified by flash chromatographyeluting from silica gel with a gradient of 0-20% MeOH in DCM to afford0.22 g of compound 73 as a white solid. MS: (+) m/z 223.3 (M+1-Boc).

Compound 74. A mixture of compound 73 (0.2 g, 0.620 mmol) and 4N HCl in1,4-dioxane (4 mL, 0.620 mmol) was stirred at RT for 1 h. The solventwas evaporated. The white solid compound 74 was washed with hexanestwice. MS: (+) m/z 223.3 (M+1).

Compound 75a. DIEA (3.23 μL, 0.019 mmol) was added to a solution ofcompound 71a (10 mg, 0.019 mmol) and HATU (7.05 mg, 0.019 mmol) in DMF(0.5 mL), adjusting pH to 8-9. After the reaction mixture was stirred atRT for 20 min, DIEA (3.23 μL, 0.019 mmol) and compound 74 (5.35 mg,0.024 mmol) in DMF (0.5 mL) were added, adjusting the pH to 8-9. Afterthe reaction mixture was stirred at RT for 1 h, LCMS showed the reactionwent to completion. The reaction was quenched by addition of 10 mL of1:1 (v/v) mixture of acetonitrile and water containing 0.1% TFA. Thecrude product was purified by preparative HPLC to afford 6.0 mg ofcompound 75a as a white solid. MS: (+) m/z 744.4 (M+1). Compound 75a isalso referred to hereinabove as compound (I-10).

Compound 75a had an EC₅₀ of 75.8 nM against N87 gastric cancer cells,using an ATP luminescence assay.

Compound 75b. DIEA (2.83 μL, 0.016 mmol) was added to a solution ofcompound 71b (10 mg, 0.016 mmol) and HATU (6.17 mg, 0.016 mmol) in DMF(0.5 mL), adjusting pH to 8-9. After the reaction mixture was stirred atRT for 20 min, DIEA (2.83 μL, 0.016 mmol) and compound 74 (4.69 mg,0.021 mmol) in DMF (0.5 mL) were added, adjusting the pH to 8-9. Afterthe reaction mixture was stirred at RT for 1 h, LCMS showed the reactionwent to completion. The reaction was quenched by addition of 10 mL of1:1 (v/v) mixture of acetonitrile and water containing 0.1% TFA. Thecrude product was purified by preparative HPLC to afford 7.6 mg ofcompound 75b as a white solid. MS: (+) m/z 820.4 (M+1). Compound 75b isalso referred to hereinabove as compound (I-11).

Compound 75b had an EC₅₀ of 0.39 nM against N87 gastric cancer cells,using an ATP luminescence assay.

Compound 75c. DIEA (3.07 μL, 0.018 mmol) was added to a solution ofcompound 71c (10 mg, 0.018 mmol) and HATU (6.70 mg, 0.018 mmol) in DMF(0.5 mL), adjusting the pH to 8-9. After the reaction mixture wasstirred at RT for 20 min, DIEA (3.07 μL, 0.018 mmol) and compound 74(5.09 mg, 0.023 mmol) in DMF (0.5 mL) were added, adjusting the pH to8-9. After the reaction mixture was stirred at RT for 1 h, LCMS showedthe reaction went to completion. The reaction was quenched by additionof 10 mL of 1:1 (v/v) mixture of acetonitrile and water containing 0.1%TFA. The crude product was purified by preparative HPLC to afford 7.6 mgof compound 75c as a white solid. MS: (+) m/z 772.5 (M+1). Compound 75cis also referred to hereinabove as compound (I-12).

Compound 75c had an EC₅₀ of 5.4 nM against N87 gastric cancer cells,using an ATP luminescence assay.

Example 19 Compound 80

This example describes the synthesis of a compound 80 suitable forconjugation via “click” chemistry, having a cyclooctyne group that canreact with an azide group in a partner molecule. The correspondingsynthetic scheme is shown in FIG. 23.

Compound 77. DCC (22.40 mg, 0.109 mmol) was added to a solution ofDBCO-PEG4-Acid 76 (50 mg, 0.090 mmol, purchased from Click ChemistryTools, Scottsdale, Ariz.) and N-hydroxysuccinimide (NHS, 20.83 mg, 0.181mmol) in DCM (1 mL) at RT. The reaction mixture was stirred at RTovernight. The solid was filtered off, and the filtrate concentrated toafford compound 77. MS: (+) m/z 650.3 (M+1).

Compound 78. DIEA was added to a solution of compound 77 (58.8 mg, 0.091mmol) and compound 21 (57.6 mg, 0.100 mmol) in DMF (1 mL) at RT,adjusting the pH to 8-9. After the reaction mixture was stirred at RTfor 1 h, LCMS showed the reaction went to completion. The solvent wasevaporated. The crude product was purified by preparative HPLC to afford20 mg of compound 78 as a white solid. MS: (+) m/z 1113.6 (M+1).

Compound 79. TFA (0.5 mL, 6.53 mmol) was added to a mixture of compound78 (17.2 mg, 0.015 mmol) in DCM (1 mL) at RT. After the reaction mixturewas stirred at RT for 1 h, LCMS showed the reaction went to completion.The solvent was evaporated to afford compound 79. MS: (+) m/z 1013.5(M+1).

Compound 80. DIEA (2.96 μL, 0.017 mmol) was added to a solution ofcompound 9 (8.55 mg, 0.015 mmol) and HATU (5.87 mg, 0.015 mmol) in DMF(0.4 mL). The pH of the reaction mixture was adjusted to 8-9. After thereaction mixture was stirred at RT for 20 min, compound 79 (15.65 mg,0.015 mmol) in DMF (1 mL) and DIEA (2.96 μL, 0.017 mmol) were added. ThepH of the reaction mixture was adjusted to 8-9. After the reactionmixture was stirred at RT for another 20 min, LCMS showed the reactionwent to completion. The solvent was evaporated. The crude product wasredissolved in 1 mL of DMSO, and purified by preparative HPLC to affordcompound 80 as a white solid. MS: (+) m/z 775.0 (M/2+1). Compound 80 isalso referred to hereinabove as compound (III-12).

A conjugate of compound 80 and seven variants of anti-glypican 3antibody 4A6 modified to have an azide group at various locationsexhibited EC₅₀'s of 1.4, 0.17, 0.13, 0.22, 0.14, 0.064, and 0.25 nMagainst N87 gastric cancer cells, using a ³H thymidine incorporationassay.

The preparation and characterization of antibody 4A6 is described inTerrette et al. 2010, the disclosure of which is incorporated herein byreference. The V_(H) CDR1, CDR2, and CDR3 and V_(K) CDR1, CDR2, and CDR3sequences for antibody 4A6 are given in SEQ ID NO:17, SEQ ID NO:18, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, respectively.The variable region V_(H) and V_(K) sequences are provided in SEQ IDNO:23 and SEQ ID NO:24, respectively.

In one embodiment, this invention provides a conjugate of compound 80and a polypeptide, preferably an antibody, modified to include an azidegroup.

Example 20 Compound 88

This example describes the synthesis of compound 88, which has an alkylprimary amine functionality that can be used for conjugation. Referenceis made to the synthetic scheme of FIG. 24.

Compound 82. A mixture of tert-butyl1-amino-3,6,9,12-tetraoxapentadecan-15-oate 54 (0.285 g, 0.888 mmol,purchased from VWR; see also Example 8) and 2,5-dioxo-pyrrolidin-1-yl6-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)hexanoate 81 (0.4 g, 0.888mmol, purchased from Chem-Impex) in DMF (3 mL) was stirred at RT for 2h. The solvent was evaporated. The crude product was purified by flashchromatography eluting from silica gel with a gradient of 0-10% MeOH inDCM to afford 0.4 g of compound 82 as a white solid. MS: (+) m/z 657.4(M+1).

Compound 83. A mixture of compound 82 (0.258 g, 0.393 mmol) in TFA (2mL, 0.393 mmol) was stirred at RT for 1 h. The solvent was evaporated.Compound 83 (white solid) was washed twice with hexanes and used for thenext step reaction without further purification.

Compound 84. DCC (0.162 g, 0.786 mmol) was added to a mixture ofcompound 83 (0.236 g, 0.393 mmol) and NHS (0.090 g, 0.786 mmol) in DCM(5 mL) at RT. After the reaction mixture was stirred at RT overnight,the solid was filtered off, and the filtrate concentrated. The crudeproduct was purified by flash chromatography eluting from silica gelwith a gradient of 0-100% EtOAc in hexanes to afford 0.195 g of compound84 as a colorless oil. MS: (+) m/z 698.3 (M+1).

Compound 85. DIEA was added to a solution of compound 21 (0.162 g, 0.279mmol) and compound 84 (0.195 g, 0.279 mmol) in DMF (1.5 mL) at RT. Afterthe reaction mixture was stirred at RT for 1 h, the reaction wasquenched by addition of 6 mL of 1:1 mixture of acetonitrile and watercontaining 0.1% TFA. The crude product was purified by preparative HPLCto afford 0.292 g of compound 85 as a white solid. MS: (+) m/z 1161.6(M+1).

Compound 86. TFA (0.5 mL) was added to a mixture of compound 85 (22.1mg, 0.019 mmol) in DCM (1 mL) at RT. After the reaction mixture wasstirred at RT for 20 min, the solvent was evaporated to afford compound86. MS: (+) m/z 1061.6 (M+1).

Compound 87. DIEA was added to a solution of compound 9 (10.53 mg, 0.019mmol) and HATU (7.23 mg, 0.019 mmol) in DMF (0.4 mL). The pH of thereaction mixture was adjusted to 8-9. After the reaction mixture wasstirred at RT for 10 min, compound 86 (20.19 mg, 0.019 mmol) in DMF (1mL) and DIEA were added. The pH of the reaction solution was adjusted to8-9. After the reaction mixture was stirred at RT for 10 min, LCMSshowed the reaction went to completion. The reaction was quenched byaddition of 10 mL of a 1:1 (v/v) mixture of water (0.1% TFA) andacetonitrile. The crude product was purified by preparative HPLC toafford 27.3 mg of compound 87 as a white solid. MS: (+) m/z 799.1(M/2+1).

Compound 88. Piperidine was added to a solution of compound 87 (27.3 mg,0.017 mmol) in DMF (2 mL) at RT, adjusting the pH to 9-10. After thereaction mixture was stirred at RT for 1 h, the reaction was quenched byaddition of 6 mL of a 1:1 mixture of acetonitrile and water containing0.1% TFA. The crude product was purified by preparative HPLC to afford22.5 mg of compound 88 as a white solid. MS: (+) m/z 688.1 (M/2+1).Compound 88 is also referred to hereinabove as compound (III-13).

In one embodiment, there is provided a conjugate in which compound 88 isconjugated to a polypeptide, which preferably is an antibody, via amideformation between the alkyl primary amine of compound 88 and a carboxylgroup on a side chain residue of an amino acid—preferably glutamicacid—in the polypeptide. This invention also provides a method of makingsuch a conjugate, comprising combining compound 88 and the polypeptidein the presence of the enzyme transglutaminase.

The foregoing detailed description of the invention includes passagesthat are chiefly or exclusively concerned with particular parts oraspects of the invention. It is to be understood that this is forclarity and convenience, that a particular feature may be relevant inmore than just the passage in which it is disclosed, and that thedisclosure herein includes all the appropriate combinations ofinformation found in the different passages. Similarly, although thevarious figures and descriptions herein relate to specific embodimentsof the invention, it is to be understood that where a specific featureis disclosed in the context of a particular figure or embodiment, suchfeature can also be used, to the extent appropriate, in the context ofanother figure or embodiment, in combination with another feature, or inthe invention in general.

Further, while the present invention has been particularly described interms of certain preferred embodiments, the invention is not limited tosuch preferred embodiments. Rather, the scope of the invention isdefined by the appended claims.

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Table Of Sequences

The following Table 3 summarizes the descriptions of the sequencesdisclosed in this application.

TABLE 3 Sequence Summary SEQ ID NO: SEQUENCE DESCRIPTION 1 6A4 V_(H)CDR1 amino acid 2 6A4 V_(H) CDR2 amino acid 3 6A4 V_(H) CDR3 amino acid4 6A4 V_(K) CDR1 amino acid 5 6A4 V_(K) CDR2 amino acid 6 6A4 V_(K) CDR3amino acid 7 6A4 V_(H) amino acid 8 6A4 V_(K) amino acid 9 1F4 V_(H)CDR1 amino acid 10 1F4 V_(H) CDR2 amino acid 11 1F4 V_(H) CDR3 aminoacid 12 1F4 V_(K) CDR1 amino acid 13 1F4 V_(K) CDR2 amino acid 14 1F4V_(K) CDR3 amino acid 15 1F4 V_(H) amino acid 16 1F4 V_(K) amino acid 174A6 V_(H) CDR1 amino acid 18 4A6 V_(H) CDR2 amino acid 19 4A6 V_(H) CDR3amino acid 20 4A6 V_(K) CDR1 amino acid 21 4A6 V_(K) CDR2 amino acid 224A6 V_(K) CDR3 amino acid 23 4A6 V_(H) amino acid 24 4A6 V_(K) aminoacid 25 Modified antibody constant region 26 Modified antibody constantregion

What is claimed is:
 1. A compound having a structure represented byformula (I)

wherein R¹ is H, unsubstituted or substituted C₁-C₁₀ alkyl,unsubstituted or substituted C₂-C₁₀ alkenyl, unsubstituted orsubstituted C₂-C₁₀ alkynyl, unsubstituted or substituted aryl,unsubstituted or substituted heteroaryl, unsubstituted or substituted(CH₂)₁₋₂O(C₁-C₁₀ alkyl), unsubstituted or substituted (CH₂)₁₋₂O(C₂-C₁₀alkenyl), unsubstituted or substituted (CH₂)₁₋₂O(C₂-C₁₀ alkynyl),(CH₂)₁₋₂OC(═O)(C₁-C₁₀ alkyl), unsubstituted or substituted(CH₂)₁₋₂OC(═O)(C₂-C₁₀ alkenyl), unsubstituted or substituted(CH₂)₁₋₂OC(═O)(C₂-C₁₀ alkynyl), unsubstituted or substitutedC(═O)(C₁-C₁₀ alkyl), unsubstituted or substituted C(═O)(C₂-C₁₀ alkenyl),unsubstituted or substituted C(═O)(C₂-C₁₀ alkynyl), unsubstituted orsubstituted cycloaliphatic, unsubstituted or substitutedheterocycloaliphatic, unsubstituted or substituted arylalkyl, orunsubstituted or substituted alkylaryl; R² is H, unsubstituted orsubstituted C₁-C₁₀ alkyl, unsubstituted or substituted C₂-C₁₀ alkenyl,unsubstituted or substituted C₂-C₁₀ alkynyl, unsubstituted orsubstituted aryl, unsubstituted or substituted heteroaryl, unsubstitutedor substituted (CH₂)₁₋₂O(C₁-C₁₀ alkyl), unsubstituted or substituted(CH₂)₁₋₂O(C₂-C₁₀ alkenyl), unsubstituted or substituted (CH₂)₁₋₂O(C₂-C₁₀alkynyl), (CH₂)₁₋₂OC(═O)(C₁-C₁₀ alkyl), unsubstituted or substituted(CH₂)₁₋₂OC(═O)(C₂-C₁₀ alkenyl), unsubstituted or substituted(CH₂)₁₋₂OC(═O)(C₂-C₁₀ alkynyl), unsubstituted or substitutedC(═O)(C₁-C₁₀ alkyl), unsubstituted or substituted C(═O)(C₂-C₁₀ alkenyl),unsubstituted or substituted C(═O)(C₂-C₁₀ alkynyl), unsubstituted orsubstituted cycloaliphatic, unsubstituted or substitutedheterocycloaliphatic, unsubstituted or substituted arylalkyl,unsubstituted or substituted alkylaryl, or

wherein each R^(2a) is independently H, NH₂, NHMe, Cl, F, Me, Et, or CN;R^(3a) and R^(3b) are independently H, C₁-C₅ alkyl, CH₂(C₅-C₆cycloalkyl), CH₂C₆H₅, C₆H5, or CH₂CH₂OH; R⁴ is

wherein R^(4a) is H or C₁-C₃ alkyl; and Y is H, OH, Cl, F, CN, Me, Et,NO₂, or NH₂; R⁵ is H, C₁-C₅ alkyl, C₂-C₅ alkenyl, C₂-C₅ alkynyl,CO(C₁-C₅ alkyl), CO(C₂-C₅ alkenyl), or CO(C₂-C₅ alkynyl); W is O or S;and n is 0, 1, or 2; or a pharmaceutically acceptable salt thereof.
 2. Acompound according to claim 1, having a structure represented by formula(Ia)

wherein Y is H or NO₂; R^(4a) is H, Me, or Et; and R^(3a) and R^(3b) areindependently H, Me, or Et.
 3. A compound according to claim 1, having astructure represented by formula (Ib):

where R^(4a) is H, Me, or Et; R^(3a) and R^(3b) are independently H, Me,and Et; and R⁶ is C₁-C₅ alkyl, CH₂OC(═O)C₁-C₅ alkyl, or (CH₂)₁₋₂C₆H₅. 4.A compound according to claim 3, wherein one of R^(3a) and R^(3b) is Hand the other is Me.
 5. A compound according to claim 3, having astructure represented by formula (Ib′):

where R^(4a) is H, Me, or Et and R⁶ is Me or n-Pr.
 6. A conjugatecomprising a compound according to claim 1 covalently linked to atargeting moiety that specifically or preferentially binds to a tumorassociated antigen.
 7. A conjugate according to claim 6, having astructure represented by formula (II-1′):

where R⁶ is Me or n-Pr and Ab is an antibody.
 8. A conjugate accordingto claim 7, wherein the antibody is an anti-CD70, anti-mesothelin, oranti-glypican 3 antibody.
 9. A conjugate according to claim 6, having astructure represented by formula (II)[D(X^(D))_(a)C(X^(Z))_(b)]_(m)Z  (II) wherein Z is a targeting moiety;X^(D) is a first spacer moiety; X^(Z) is a second spacer moiety; C is acleavable group; subscripts a and b are independently 0 or 1; subscriptm is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and D is according to formula(D-a)

or formula (D-b)

wherein Y is H or NO₂; R^(4a) is H, Me, or Et; R^(3a) and R^(3b) areindependently H, Me, or Et; and R⁶ is C₁-C₅ alkyl, CH₂OC(═O)C₁-C₅ alkyl,or (CH₂)₁₋₂C₆H₅; or a pharmaceutically acceptable salt thereof.
 10. Aconjugate according to claim 9, wherein Z is an antibody.
 11. Adrug-linker compound having a structure according to formula (III)D-(X^(D))_(a)C(X^(Z))_(b)—R³¹  (III) wherein R³¹ is a reactivefunctional group; X^(D) is a first spacer moiety; X^(Z) is a secondspacer moiety; C is a cleavable group; subscripts a and b areindependently 0 or 1; subscript m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;and D is according to formula (D-a)

or formula (D-b)

wherein Y is H or NO₂; R^(4a) is H, Me, or Et; and R^(3a) and R^(3b) areindependently H, Me, or Et; and R⁶ is C₁-C₅ alkyl, CH₂OC(═O)C₁-C₅ alkyl,or (CH₂)₁₋₂C₆H₅; or a pharmaceutically acceptable salt thereof.
 12. Adrug-linker compound according to claim 11, wherein R³¹ is —NH₂, —OH,—CO₂H, —SH, maleimido, cyclooctyne, azido, hydroxylamino, orN-hydroxysuccinimido.
 13. A drug-linker compound according to claim 11,having a structure represented by formula (III-a):

where R^(3a) and R^(3b) are independently H, Me, or Et; R⁶ is Me, Et, orn-Pr; AA^(a) and each AA^(b) are independently selected from the groupconsisting of alanine, β-alanine, γ-aminobutyric acid, arginine,asparagine, aspartic acid, γ-carboxyglutamic acid, citrulline, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,lysine, methionine, norleucine, norvaline, ornithine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, and valine; p is 1, 2,3, or 4; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; r is 1, 2, 3, 4, or 5; sis 0 or 1; and R³¹ is selected from the group consisting of


14. A drug-linker compound according to claim 11, having a structurerepresented by formula (III-b):

where R⁶ is Me or n-Pr; q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; r is 1,2, 3, 4, or 5; s is 0 or 1; and R³¹ is selected from the groupconsisting of


15. A pharmaceutical composition comprising a compound according toclaim 1, or a conjugate thereof with a targeting moiety, and apharmaceutically acceptable carrier.
 16. A pharmaceutical compositionaccording to claim 15, wherein the compound according to claim 1 isconjugated to a targeting moiety that is an antibody.
 17. A compoundaccording to claim 1, having a structure represented by formula (I-9):


18. A conjugate according to claim 7, wherein the antibody is ananti-mesothelin antibody and R⁶ is n-Pr.
 19. A drug-linker compoundaccording to claim 11, having a structure represented by formula(III-8):